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Xiaosheng Zhao, Chaorong Meng, , Zaifu Yang, Xue-Jun Pan
Published: 16 January 2022
Abstract:
Magnolia grandiflora is a widely cultivated ornamental tree in China. In June 2020, a leaf blight disease was observed on M. grandiflora in Guizhou University (26° 44' 57'' N, 106° 65' 94'' E) in Guiyang, China. The initial symptoms on leaves were expanding round necrotic lesions with a grey center and dark brown edge, and twigs were withered when the disease was serious. Of the 100 plants surveyed 65% had symptoms. To isolate the potential causal pathogen, diseased leaves were collected from an M. grandiflora tree at Guizhou University. Isolations from made form the junction between healthy and symptomatic tissue and disinfested by immersing in 75% ethanol for 30 seconds, 3% NaOCl for 2 minutes, and then washed 3 times in sterile distilled water. Symptomatic tissue was then plated on potato dextrose agar (PDA) and incubated at 25ºC with 12-hour light for 3–5 days. Three isolates (GUCC 21235.1, GUCC 21235.2 and GUCC 21235.3) were obtained. Colonies on PDA after 7 d were dark brown, pycnidia embedded in the mydelium were dark brown to black, single and separated. Conidiophores were transparent measuring 7–12.5 × 2.5–4.5 µm (mean = 9.5 × 3.6 µm, n = 30) in length. Conidia were transparent becoming brown when mature with a diaphragm, with round ends measuring, 21–27 × 10–15 µm (mean = 23.6 × 12.6 µm, n = 30). To confirm the pathogen by molecular characterization, four genes or DNA fragments, ITS, LSU, tef1 and β-tubulin, were amplified using the following primer pairs: ITS4-F/ ITS5-R (White et al., 1990), LR0R/ LR5 (Rehner & Samuels, 1994), EF1-688F/ EF1-986R (Carbone & Kohn, 1999) and Bt2a/ Bt2b (O'Donnell & Cigelnik, 1997). The sequences of four PCR fragments of GUCC 21235.1 were deposited in GenBank, and the accession numbers were MZ519778 (ITS), MZ520367 (LSU), MZ508428 (tef1) and MZ542354 (β-tubulin). Bayesian inference was performed based on a concatenated dataset of ITS, LSU, tef1 and β-tubulin gene using MrBayes 3.2.10, and the isolates GUCC 21235.1 formed a single clade with the reference isolates of Diplodia mutila (Diplodia mutila strain CBS 112553). BLASTn analysis indicated that the sequences of ITS, LSU, tef1 and β-tubulin revealed 100% (546/546 nucleotides), 99.82% (568/569 nucleotides), 100% (302/302 nucleotides), and 100% (437/437 nucleotides) similarity with that of D. mutila in GenBank (AY259093, AY928049, AY573219 and DQ458850), respectively. For confirmation of the pathogenicity of this fungus, a conidial suspension (1×105 conidia mL-1) was prepared from GUCC 21235.1, and healthy leaves of M. grandiflora trees were surface-disinfested by 75% ethanol, rinsed with sterilized distilled water and dried by absorbent paper. Small pieces of filter paper (5 mm ×5 mm), dipped with 20 µL conidial suspension (1×105 conidia mL-1) or sterilized distilled water (as control), were placed on the bottom-left of the leaves for inoculation. Then the leaves were sprayed with sterile distilled water, wrapped with a plastic film and tin foil successively to maintain high humidity in the dark dark. After 36 h, the plastic film and tin foil on the leaves was removed, and the leaves were sprayed with distilled water three times each day at natural condition (average temperature was about 25 °C, 14 h light/10 h dark). After 10 days of inoculation, the same leaf blight began to appear on the leaves inoculated with conidial suspension. No lesion was appeared on the control leaves. The fungus was re-isolated from the symptomatic tissue. Based on the morphological information and molecular characterization, the isolate GUCC 21235.1 is D. mutila. Previous reports indicated that D. mutila infects a broad host range and gives rise to a canker disease of olive, apple and jujube (Úrbez-Torres et al., 2013; Úrbez-Torres et al., 2016; Feng et al., 2019). This is the first report of leaf blight on M. grandiflora caused by D. mutila in China.
Published: 14 January 2022
Abstract:
Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter americanus’ (CLam) are associated with HLB in Brazil, but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid – ACP) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison to CLas, suggesting a possible role on host-interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides aiming to evaluate the acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5 and Pep6 in artificial diet significantly reduced the acquisition of CLas, while increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas and sweet orange plants stably absorbed and maintained this peptide for as long as three months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.
Published: 14 January 2022
Abstract:
‘Candidatus Liberibacter asiaticus’ (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.
Published: 14 January 2022
Abstract:
Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.
John S. Ramsey, , Jaclyn E. Mahoney, , Richard Johnson, David O. Igwe, Theodore W. Thannhauser, , David G. Hall,
Published: 14 January 2022
Abstract:
The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.
Published: 14 January 2022
Abstract:
Citrus greening, or Huanglongbing (HLB), currently is the most destructive disease of citrus. HLB disease is putatively caused by the phloem-restricted α-proteobacterium, ‛Candidatus Liberibacter asiaticus’. This bacterium is primarily transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Most animal pathogens are considered pathogenic to their insect vectors, whereas the relationships between plant pathogens and their insect vectors are variable. Lately, the relationship of ‛Ca. L. asiaticus’ with its insect vector, D. citri was well investigated at the molecular, biochemical, and biological levels in many studies. Herein, the findings concerning this relationship are discussed and molecular features of the acquisition of ‛Ca. L. asiaticus’ from the plant host and its growth and circulation within D. citri, as well as its transmission to plants, are presented. In addition, the effects of ‛Ca. L. asiaticus’ on the energy metabolism (respiration, TCA cycle, the ATP production), metabolic pathways, immune system, endosymbionts, and detoxification enzymes of D. citri are discussed together with other impacts such as shorter lifespan, altered feeding behavior, and higher fecundity. Overall, although ‛Ca. L. asiaticus’ has significant negative effects on its insect vector, it increases its vector fitness, indicating that it develops a mutualistic relationship with its vector. This review will help in understanding the specific interactions between ‛Ca. L. asiaticus’ and its psyllid vector in order to design innovative management strategies.
Published: 14 January 2022
Abstract:
Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development.
Published: 14 January 2022
Abstract:
Candidatus’ Liberibacter asiaticus is associated with the devastating citrus disease Huanglongbing. It is transmitted by grafting infected material to healthy plants and by the feeding of the Asian citrus psyllid (Diaphorina citri). Previously, we demonstrated that a metabolomics approach using proton-Nuclear Magnetic Resonance spectroscopy discriminates healthy from diseased plants via grafting. The present work assessed the capability of this technology in discriminating healthy and diseased plants when the bacterium is vectored by psyllids. One-year old greenhouse-grown ‘Lisbon’ lemon trees were exposed to either carrier psyllids (Exposed, n = 10), or psyllids that were free of Liberibacter asiaticus (Control, n = 6). Leaf metabolites were tracked for 1 year and disease diagnosis was made using quantitative polymerase chain reaction. Overall, 31 water-soluble metabolites were quantified in leaves, including 4 sugars and 12 amino acids. Analysis via non-metric multidimensional scaling and principal components analysis revealed significant differences between the leaf metabolome of Control vs. infected trees beginning at 8 weeks post-exposure, including alterations in glucose and quinic acid concentrations. These findings provide a longitudinal overview of the metabolic effects of HLB during the early phases of disease, and confirm previous experimental work demonstrating that infection elicits changes in the leaf metabolome that enables discrimination between healthy and infected plants. Here we demonstrate that the mode of inoculation (i.e. graft- vs. psyllid-) results in a similar pathology.
, , Dekang Wang, , Wensi Li, , Zhiwen Feng,
Published: 14 January 2022
Abstract:
Pectobacterium spp. and Dickeya spp. cause blackleg and soft rot on potato worldwide (Charkowski, 2018). Potato plants (cv. Favorita or Jizhang 8#) with blackleg symptoms (vascular browning of crown stems, Fig. S1) were observed in the field in Zhangjiakou, Hebei province in 2018, and in Ningde, Fujian Province in 2019, in China. The disease incidence was around 50% and 10% in Zhangjiakou (5 ha) and Ningde (4 ha), respectively. Diseased plants (3 from each site) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, 4 bacterial colonies in total which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Two colony sequences that showed more than 99% sequence identity to Pectobacterium punjabense type strain SS95 (MH249622) were submitted to the GenBank ( accession numbers: OK510280, MT242589). Additionally, six housekeeping genes proA (OK546205, OK546199), gyrA (OK546206, OK546200), icdA (OK546207, OK546201), mdh (OK546208, OK546202), gapA (OK546209, OK546203), and rpoS (OK546210, OK546204) of these two isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with MH249622T . Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 2 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings and potato tubers (cv. Favorita) by injecting 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems or the tubers and kept at 100% humidity and 21°C for 1 day. Four days after inoculation, the infected area of the inoculated seedlings rotten and turned black, while the controls were symptomless (Fig. S4). Two days after inoculation, the infected tubers rotten and turned black, while the controls were symptomless (Fig. S4). Bacterial colonies were reisolated from these symptomatic tissues and identified using the same methods described above. Blackleg on potato plants or soft rot on potato has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium parmentieri, Pectobacterium polaris in China (Zhao et al., 2018; Cao et al., 2021; Wang et al., 2021). To our knowledge, this is the first report of blackleg/soft rot of potato caused by Pectobacterium punjabense in China. We believe that this report will draw attention to the management of this pathogen in China.
, NingXing Zhou, RamanDeep Bamrah, ,
Published: 13 January 2022
Abstract:
‘Candidatus Liberibacter’ species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of ‘Ca. Liberibacter’ in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify ‘Ca. Liberibacter’ in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a ‘Ca. Liberibacter solanacearum’ sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that ‘Ca. Liberibacter solanacearum’ species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by ‘Ca. Liberibacter’ pathogens, and this possibility warrants further study.
Published: 13 January 2022
Abstract:
Boxwood is one of the most common and widely planted perennial ornamentals in both home gardens and commercial landscapes. Recently reported boxwood dieback, a fungal disease caused by Colletotrichum theobromicola, has been spreading at an alarming rate within the U.S. Boxwood breeders, nursery growers, and landscape professionals have shown great concerns regarding the lack of effective management practices. Therefore, the primary objectives of this study were to devise effective disease management strategies including screening cultivars to determine their susceptibility to boxwood dieback and screening various fungicides to determine their effectiveness in managing the disease. Host range studies were conducted by screening a wide variety of boxwood cultivars under greenhouse conditions. Although, boxwood cultivar ‘Little Missy’ showed much delayed symptom expression as compared to rest of the cultivars but none of the 11 cultivars were found to be resistance to boxwood dieback. In vitro screening of nine fungicides was conducted to determine mycelial growth as well as spore germination inhibition of eight isolates of C. theobromicola collected from eight states in the U.S. Of the nine fungicides, difenoconazole+pydiflumetofen showed maximum mycelial growth and spore germination inhibition at 1 ppm active ingredient followed by fluxapyroxad+pyraclostrobin, and pyraclostrobin+boscalid at 5 ppm active ingredient. Azoxystrobin+benzovindiflupyr significantly inhibited mycelial growth at 1 ppm but reduced spore germination at 10 ppm active ingredient. This study provides the boxwood industry professionals with critical and applied information pertaining to host susceptibility and fungicide efficacy to effectively mitigate boxwood dieback and to reduce its further spread.
, Diann Achor,
Published: 13 January 2022
Abstract:
‘Candidatus Liberibacter asiaticus’ (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.
, Sun Wei, Huizhu Yuan, Xiaojing Yan
Published: 13 January 2022
Abstract:
Gerbera daisy, Gerbera jamesonii H. Bolus ex. Hooker, is an important flower grown globally. In September 2020, gerbera seedlings in a greenhouse farm in the region of Fujian, China, developed symptoms of severe wilting and stunting. The main stem exhibited reddish to light brown vascular discoloration. Approximately 30% of the 60,000 plants showed symptoms. To isolate the causal agent, necrotic tissue pieces (3×3 mm) from the symptomatic stem were surface-disinfected with 1% NaClO for 1 min and washed three times with sterile water. The disinfected pieces were dried and placed on potato dextrose agar (PDA) at 25°C in the darkness for 4 days inside a dark chamber. Reddish-white and floccose mycelia developed on PDA after 3 days incubation. Ten single-spored isolates were identified as Fusarium kyushuense based on morphological features (Aoki & O'Donnell, 1998). Hyaline and straight or slightly curved macroconodia were observed with 3 to 5 septate, 24.5 - 46.6 × 3.6 - 5.7 μm (n = 100). Microconidia were ellipsoidal to clavate, 0 to 1 septate, and 6.3 to 19.5 × 3.2 to 5.3 μm (n = 100). No chlamydospores were observed. In order to validate this result, partial RNA polymerase second largest subunit (RPB2) combined with translation elongation factor (EF-1α) gene regions were amplified and sequenced from three isolates with primers 5f2/11ar (Liu et al., 1999) and primers EF1/EF2 (Geiser et al. 2004), respectively. Fusarium MLST analysis showed that the RPB2 (Genbank accession No. MZ130468, No. MZ130469, No. MZ130470) matched 99.72% (MH582170) to F. kyushuense, and the EF-1α (MZ130471, MZ130472, MZ130473) matched 99.84% (MH582297) to F. kyushuense in the Fusarium MLST. Besides, a phylogenetic analysis was conducted using the neighbor-joining algorithm based on the RPB2 and EF-1α gene sequences. The isolates clustered with F. kyushuense. To assess pathogenicity, the three molecularly identified isolates were used. The isolates were grown on carboxylmethyl cellulose (CMC) medium (carboxymethyl-cellulose (Sigma C-4888) 15.0 gram, NH4NO3 1.0 gram, KH2PO4 monobasic 1.0 gram, MgSO4·7H2O 0.5 gram, yeast extract 1.0 gram, distilled water filled to 1.0 liter) for sporulation. The roots of 12 healthy 30-day-old gerbera plants were inoculated by treating them with 10 mL of conidia suspension (1×106 conidial/mL). A group of 12 seedlings of the same age was treated with sterile water to serve as the control. Plants were grown in a glasshouse at 23 °C, relative humidity >70%, and 16 h light per day. Typical symptoms of wilt and discoloration of the vascular system in roots and stems developed within 10 days. Uninoculated plants remained healthy. Isolates were consistently re-isolated from the symptomatic stem and the recovered isolates were identified as F. kyushuense by amplifing the EF-1α gene. The assays were conducted twice. F. kyushuense has been reported to cause wilt and rot of tobacco (Wang et al., 2013), maize ears (Wang et al., 2014) and rice (Zhao et al., 2007) in China. To the best of our knowledge, this is the first report of F. kyushuense causing stem and root wilt on G. jamesonii. The disease must be considered in existing management practices.
Bong Nam Chung, , Ju-Yeon Yoon, In-Sook Cho
Published: 13 January 2022
Abstract:
Cnidium officinale is a perennial plant in the family Apiaceae. It is native to China and cultivated in China, Japan, and Korea for its roots for medicinal purposes. In August 2019, 63 C. officinale plants showing symptoms of vein chlorosis, yellowing and chlorotic spots (Supplementary Fig. 1) were collected from commercial farms in Bonghwa and Youngyang, Gyeongsangbuk-do, South Korea. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to confirm the presence of apple stem grooving virus (ASGV), cnidium vein yellowing virus 1, cnidium vein yellowing virus 2, lychnis mottle virus, and Cnidium virus X with specific primers (Supplementary Table 1). Forty-one out of the sixty-three samples were positive for ASGV in mixed infection with one or more of the other four viruses. Nicotiana benthamiana plants mechanically inoculated with the crude sap of one of the ASGV-infected C. officinale plants showed mosaic symptom on upper leaves 10 days post inoculation (dpi). Infection was confirmed by RT-PCR and Sanger sequencing. N. benthamiana plants systemically infected with ASGV-CO-kr1 isolate alone were used for subsequent sequencing and host range test. Twenty-day old seedlings of 23 species of plants (two to 14 species for each family) from the families Solanaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and Amaranthaceae (Supplementary Table 2) were mechanically inoculated with sap of ASGV-CO-kr1-infected N. benthamiana plants. ASGV-CO-kr1 infected all tested 23 species as confirmed by symptomology, RT-PCR, and Sanger sequencing at 10 to 20 dpi. The MP and CP genes of ASGV-CO-kr1 were amplified by RT-PCR with specific primers 4300-4325F/5642-5666R and 5592-5612F/6475-6499R, respectively (Supplementary Table 1). The amplicons were cloned and sequenced (GenBank accession numbers: MP = MW889883 and CP = MW889884). Multiple sequence alignment using the MegAlign program in DNASTAR showed that the complete CP and MP genes of ASGV-CO-kr1 shared 89.9%-99.7% and 83.1%-99.5% identities, respectively at the nucleotide (nt) level and they shared 92.4%-99.6% and 93.8%-99.4% identities, respectively at amino acid (aa) level with corresponding sequences of 34 other ASGV isolates from various host plants and countries. Phylogenetic analysis with the Maximum Likelihood method using the MEGA X program (Kumar et al., 2018) showed that ASGV-CO-kr1 grouped with isolates Cuiguan (KR185346), BH (LC480456), and YY (LC480457) based on the CP aa sequences, while it grouped with isolates SG (LC475148) and TL101 (MH108976) based on the MP aa sequences. ASGV is known to naturally infect apples, European pear, Asian pear, citrus, apricot, cherry, kiwifruit, loquat, lily, and lotus (Clover et al., 2003; He et al., 2019; Hu et al., 2017; Liu et al., 2017; Yanase et al., 1975). To the best of our knowledge, this is the first report of the natural infection of ASGV in C. officinale. C. officinale plants are propagated by root division, so they are susceptible to infection with viruses. The result of this study is important for generating virus-free seedlings to produce C. officinale.
Published: 13 January 2022
Abstract:
Huanglongbing (HLB), or citrus greening disease, has significantly decreased citrus production all over the world. The disease management currently depends on the efficient application and adequate distribution of insecticides to reduce the density of the disease vector, the Asian citrus psyllid. Here, we use a novel fluorescent-based method to evaluate insecticide distribution in an HLB-infected citrus grove in Florida. Specifically, we evaluated six different locations within citrus trees, the top and bottom sides of leaves, the effect of application approach (tractor versus airplane), and different application rates. We found that despite the insecticide distribution being highly variable among the different locations within a tree, the top of the leaves received an average increase of 21 times more than the bottom of the leaves. Application by tractor also resulted in a 4- to 87-fold increase in insecticide coverage compared with aerial application, depending on the location in the tree and side of the leaf. When taken to context with the type of insecticide that is applied (systemic vs. contact), these results can be used to optimize a pest management strategy to effectively target psyllids and other pests while minimizing the time and money spent on insecticide application and reducing risk to the environment.
Published: 13 January 2022
Abstract:
A decade ago, shoot proliferation symptoms (witches' broom) in carrots were believed to be the cause of 'Candidatus Phytoplasma' and/or Spiroplasma infection, yet in recent years, this association appeared to have weakened and a closer association was found with the yet-unculturable, psyllid-transmitted Gram-negative bacterium, 'Candidatus Liberibacter solanacearum'. In Israel, carrots are grown throughout the year, yet shoot proliferation symptoms tend to appear only in mature plants and mostly during late spring to early summer. We hypothesized that factors such as plant age, temperature and vector load, which vary along the year, have a critical effect on symptoms development and set to examine these factors under controlled conditions. Here we show that young carrot seedlings are as prone as older plants, to develop shoot proliferation symptoms, following 'Ca. L. solanacearum' inoculation. Surprisingly, we found that the local 'Ca. L. solanacearum' haplotype was extremely sensitive to constant temperature of 30˚C, which led to a significant reduction in bacterial growth and symptoms development, compared with 18˚C which was very conducive for symptoms development. We have also found that inoculations with 10 or 20 psyllids per plant results in faster symptoms development compared with inoculations with 2 psyllids per plant, however, the disease progress rate was insignificant among the different vector loads. These data provide important insight to the effects of plant age, temperature and vector load on 'Ca. L. solanacearum' and its associated symptoms and strengthen the notion that 'Ca. L. solanacearum' is the main responsible agent for carrot witches broom in Israel.
Published: 13 January 2022
Abstract:
Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum is an important leaf spot disease in the mid-Atlantic U.S. Disease management approaches include use of resistant varieties, cultural control, and foliar fungicides. Frequent use of foliar fungicides can select for fungicide resistance within pathogen populations. Recently, the first report of quinone outside inhibitor (QoI) fungicide resistance in the U.S. was made based on a relatively small collection of P. nodorum isolates from Virginia. The objective of this study was to conduct a state-wide, two-year survey of P. nodorum populations in Virginia wheat and quantify frequencies of the target-site mutation that confers QoI resistance. A total of 318 isolates of P. nodorum were obtained from wheat collected at seven locations distributed throughout the wheat-growing regions of Virginia in 2018 and 2019. A previously designed pyrosequencing assay that detects the G143A substitution in the cytochrome b gene of P. nodorum was used to screen isolates for the presence or absence of the target site mutation. The G143A substitution was detected in all sampled fields. Among locations and years, frequencies of the mutation in P. nodorum populations ranged from 5-32% (mean = 19%). Thus, the QoI-resistance conferring G143A mutation was widespread in P. nodorum populations in Virginia and it occurred at a relatively high frequency. Results suggest that fungicides containing QoI active ingredients may not be effective for controlling SNB in Virginia and the surrounding region, and application of stand-alone QoI fungicides for disease control in wheat is not recommended.
Francisco Beluzán, , , , Paloma Abad-Campos, , Josep Armengol
Published: 12 January 2022
Abstract:
Twenty-five almond cultivars were assessed for susceptibility to Diaporthe amygdali, causal agent of twig canker and shoot blight disease. In laboratory experiments, growing twigs were inoculated with four D. amygdali isolates. Moreover, growing shoots of almond cultivars grafted onto INRA ‘GF-677’ rootstock were used in four-year field inoculations with one D. amygdali isolate. In both type of experiments, inoculum consisted of agar plugs with mycelium, which were inserted underneath the bark and the lesion lengths caused by the fungus were measured. Necrotic lesions were observed in the inoculated almond cultivars both in laboratory and field tests, confirming the susceptibility of all the evaluated cultivars to all the inoculated isolates of D. amygdali. Cultivars were grouped as susceptible or very susceptible according to a cluster analysis. The relationship between some agronomic traits and cultivar susceptibility was also investigated. Blooming and ripening times were found relevant variables to explain cultivars performance related to D. amygdali susceptibility. Late and very late blooming, and early and medium ripening cultivars were highly susceptible to D. amygdali. Our results may provide valuable information that could assist in ongoing breeding programs of this crop and additionally in the selection of cultivars for new almond plantations.
Published: 12 January 2022
Abstract:
Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 60,602,320 t, covering 588,597 ha (ISTAT 2021). Trichoderma species are widespread filamentous fungi in soil, well known and studied as biological control agents (Vinale et al., 2008). Seeds of a yellow grain hybrid (class FAO 700, 132 days) were collected in September 2020 from an experimental field located in Carmagnola (TO, Italy: GPS: 44°53'11.0"N 7°40'60.0"E) and tested with blotter test (Warham et al., 1996) to assess their phytosanitary condition. Over the 400 seeds tested, more than 50% showed rotting and development of green mycelium typical of the genus Trichoderma. Due to the high and unexpected percentage of decaying kernels, ten colonies were identified by morphological and molecular methods. Single conidia colonies of one Trichoderma (T5.1) strain were cultured on Potato Dextrose Agar (PDA) for pathogenicity tests, and on PDA and Synthetic Nutrient-Poor Agar (SNA) for morphological and molecular identification. The colonies grown on PDA and SNA showed green, abundant, cottony, and radiating aerial mycelium, and yellow pigmentation on the reverse. Colony radius after 72 h at 30°C was of 60-65 mm on PDA and of 50-55 mm on SNA. The isolates produced one cell conidia 2.8 - 3.8 µm long and 2.1 - 3.6 µm wide (n=50) on SNA. Conidiophores and phialides were lageniform to ampulliform and measured 4.5 – 9.7 µm long and 1.6 – 3.6 µm wide (n=50); the base measure 1.5 – 2.9 µm wide and the supporting cell 1.4 – 2.8 µm wide (n=50). The identity of one single-conidia strain was confirmed by sequence comparison of the internal transcribed spacer (ITS), the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (Oskiera et al., 2015). BLASTn searches of GenBank using ITS (OL691534) the partial tef-1α (OL743117) and rpb2 (OL743116) sequences of the representative isolate T5.1, revealed 100% identity for rpb2 to T. afroharzianum TRS835 (KP009149) and 100% identity for tef-1α to T. afroharzianum Z19 (KR911897). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile H2O to 1×106 CFU/ml. Twenty-five seeds were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 23°C under a seasonal day/night light cycle. Twenty primary ears were inoculated, by injection into the silk channel, with 1 ml of a conidial suspension of strain T5.1 seven days after silk channel emergence (BBCH 65) (Pfordt et al., 2020). Ears were removed four weeks after inoculation and disease severity, reaching up to 75% of the kernels of the twenty cobs, was assessed visually according to the EPPO guidelines (EPPO, 2015). Five control cobs, inoculated with 1 ml of sterile distilled water were healthy. T. afroharzianum was reisolated from kernels showing a green mold developing on their surface and identified by resequencing of tef-1α gene. T. afroharzianum has been already reported on maize in Germany and France as causal agent of ear rot of maize (Pfordt et al. 2020). Although several species of Trichoderma are known to be beneficial microorganisms, our results support other findings that report Trichoderma spp. causing ear rot on maize in tropical and subtropical areas of the world (Munkvold and White, 2016). The potential production of mycotoxins and the losses that can be caused by the pathogen during post-harvest need to be explored. To our knowledge this is the first report of T. afroharzianum as a pathogen of maize in Italy.
Molecular Plant-Microbe Interactions®; https://doi.org/10.1094/mpmi-11-21-0282-a

Abstract:
Curtobacterium flaccumfaciens complex species in the family Microbacteriaceae encompasses a group of plant pathogenic actinobacterial strains affecting annual crops and ornamental plants. The species includes five pathovars namely C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. ilicis, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae. Despite the economic importance of C. flaccumfaciens, its members have rarely been investigated for their phylogenetic relationships, molecular characteristics and virulence repertories due in part to the lack of whole genome resources. Here we present the whole genome sequence of 17 C. flaccumfaciens strains representing members of four pathovars isolated from different plant species in a diverse geographical and temporal span. The genomic data presented in this study will pave the way of research on the comparative genomics, phylogenomics and taxonomy of C. flaccumfaciens, and extend our understanding of the virulence features of the species.
Published: 12 January 2022
Abstract:
Systems of classification are important for guiding research activities and providing a common platform for discussion and investigation. One such system is assigning microbial taxa to the roles of mutualists and pathogens. Yet, there are often challenges and even inconsistencies in reports of research findings when microbial taxa display behaviors outside of these two static conditions (e.g. commensal). Over the last two decades, there has been some effort to highlight a continuum of symbiosis, wherein certain microbial taxa may exhibit mutualistic or pathogenic traits depending on environmental contexts, life stages, and plant host associations. However, gaps remain in understanding how to apply the continuum approach to host-microbe pairs across a range of environmental and ecological factors. This commentary presents an alternative framework for evaluating the continuum of symbiosis using dominant archetypes that define symbiotic ranges. We focus particularly on fungi and bacteria, though we recognize that archaea and other microeukaryotes play important roles in host-microbe interactions that may be described by this approach. This framework is centered in eco-evolutionary theory and aims to enhance communication among researchers, as well as prioritize holistic consideration of the factors shaping microbial life strategies. We discuss the influence of plant-mediated factors, habitat constraints, co-evolutionary forces, and the genetic contributions which shape different microbial lifestyles. Looking to the future, using a continuum of symbiosis paradigm will enable greater flexibility in defining the roles of target microbes and facilitate a more holistic view of the complex and dynamic relationship between microbes and plants.
, Jeff Schachterle, Emma Sweeney, , Jingyu Peng, , , ,
Published: 12 January 2022
Abstract:
Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the 1st day of opening were found to harbor large (106-107 cells / flower) populations of E. amylovora when assessed three to five days post-inoculation. However, populations inoculated on stigmas of flowers that were already open for three days did not reach 106 cells / flower, and populations inoculated on stigmas of flowers that were already open for five days never exceeded 104 cells / flower. During this study, >10-fold increases in E. amylovora stigma populations in a 24-hr time period (termed population surges) were observed on 34.8%, 20.0%, and 4.0% of possible days on 1-day, 3-day, and 5-day open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5°C and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 PM and 2:00 AM. A Pearson’s correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations where overnight temperatures either increased or remained constant, where wind speed decreased, and where relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.
Published: 12 January 2022
Abstract:
Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting water transport. The dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap due to Ralstonia abundance. However, the physiological mechanism of xylem disruption during bacterial wilt is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified spatiotemporal dynamics of xylem disruption during bacterial wilt. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged intact plant stems to quantify embolized vessels. Embolized vessels were rare, but infected plants with low bacterial populations had a non-significant trend of more vessel embolisms. To test that vessels are clogged during bacterial wilt, we imaged excised stems after brief dehydration. Most vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5x108 cfu/g stem tissue, approximately half of the vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in X-ray microCT reconstructions or from light microscopy of preserved stems. Therefore, bacterial blockage of vessels appears to be the principal cause of xylem disruption during Ralstonia wilt.
Published: 12 January 2022
Abstract:
Aspergillus flavus infects a wide range of crops, including pistachios, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on non-aflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. Aspergillus flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights, but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared to the lower canopy (mean = 47 CFU/g). Results suggest June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards, and to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.
, Shuping Tian, Nan Wu, , ,
Published: 12 January 2022
Abstract:
Southwest China has the most complex rice-growing regions in China. With great differences in topography, mainly consisting of basins and plateaus, ecological factors in above region differ greatly. In this study, bulk paddy soils collected from a long-term rice field in Chengdu (basins) and in Guiyang (plateaus) were used to study the correlation between microbial diversity and the incidence of rice bacterial diseases. Results showed that the microbial community composition in paddy soils and the microbial functional categories differed significantly between basins and plateaus. They shared more than 70% of the dominant genera (abundance > 1%), but the abundance of the dominant genera differed significantly. Functional analysis found that bulk paddy soils from Chengdu were significantly enriched in virulence factor-related genes; soils from Guiyang were enriched in biosynthesis of secondary metabolites especially antibiotics. Correspondingly, Chengdu was significantly enriched in leaf bacterial pathogens Acidovorax, Xanthomonas, and Pseudomonas. Greenhouse experiments and correlation analysis showed that soil chemical properties had a greater effect on microbial community composition and positively related with the higher incidence of rice bacterial foot rot in Guiyang, while temperature had a greater effect on soil microbial functions and positively related with the higher severity index of leaf bacterial diseases in Chengdu. Our results provide a new perspective on how differences in microbial communities in paddy soils can influence the incidence of rice bacterial diseases in areas with different topographies.
Huizheng Wang, Jinye Gao, Yang Zhao, , Wei He, Jingjing Shi,
Published: 12 January 2022
Abstract:
Oxalis corniculata L., which belongs to the family Oxalidaceae R. Br., is a very common perennial herb. It is usually planted on bare land or under the forest as landscaping plants, and the whole plant can be used for its medicinal values of clearing heat, detoxification and detumescence. In August 2019, typical symptoms of anthracnose on O. corniculata leaves were observed in the green belt on the campus of Shandong University of Technology (36.81°N, 117.99°E), Shandong Province, China. The disease incidence was above 40% by investigating more than 300 m2 of planting area. Most of O. corniculata are planted under the forest where the disease is found, mainly in the environment with high relative humidity and less ventilation. The infected leaves appeared initially as tawny oval or irregular spots, and then the lesions enlarged gradually until the leaves became dieback or wholly withered, which greatly reduced the landscape effect of O. corniculata. Diseased leaves were collected by cutting into small pieces and sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaClO) for 60 s, rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 7 days in the dark (Zhu et al. 2013). Fifteen isolates were obtained from 20 symptomatic leaves and the cultures were initially gray white, subsequently became grayish to dark green after 7 days, with copious gray aerial mycelium and black microsclerotia. Three isolates were verified by the amplification of DNA sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), histone H3 (H3) and chitin synthase (CHS1) genes, using the primer pairs GDF1/GDR1, ACT-512F/ACT-783R, CYLH3F/CYLH3R, and CHS-79F/CHS-234R (Damn et al. 2019, Fu et al. 2019, Liu et al. 2013), respectively. The sequenced genes (GenBank accession no. OK017473, OK159078, OK159076, OK159077) shared 99.62 to 100.00% nucleotide identity with the corresponding genes of Colletotrichum truncatum strain UASB-Cc-10 (GenBank accession no. KF322064.1, KF322055.1, KF322073.1, KF319059.1), respectively, which was consistent with the morphological identification (Sawant et al. 2012). Pathogenicity test was performed with six healthy O. corniculata plants infected with mycelial plugs (about 3 mm in diameter) of three C. truncatum isolates from a 5-day-old culture, while the negative controls on the same leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25 to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves appeared brown sunken spots, whereas no symptoms appeared on negative controls. The same pathogens, C. truncatum, were identified from the inoculated leaves on the basis of morphological and molecular characteristics as described above, confirming Koch’s postulates. To our knowledge, anthracnose caused by C. truncatum on O. corniculata is the first report in China. The discovery of this new disease is beneficial to the application and protection of O. corniculata, a popular landscape and medicinal plant. References: Damn, U., et al. 2019. Stud. Mycol. 92:1. https://doi.org/10.1016/j.simyco.2018.04.001 Fu, M., et al. 2019. Persoonia 42:1. https://doi.org/10.3767/persoonia.2019.42.01 Liu, F., et al. 2013. Mycologia 105:844. https://doi.org/10.3852/12-315 Sawant, I. S., et al. 2012. New Dis. Rep. 25:2. https://doi.org/10.5197/j.2044-0588.2012.025.002 Zhu, L., et al. 2013. J. Phytopathol. 161:59. https://doi.org/10.1111/jph.12019 The author(s) declare no conflict of interest. Acknowledgments: This research was financially supported by the Top Talents Program for One Case One Discussion of Shandong Province and Academy of Ecological Unmanned Farm (2019ZBXC200).
Published: 12 January 2022
Abstract:
Tar spot of corn caused by Phyllachora maydis has recently led to significant yield losses in the eastern corn belt of the Midwestern United States. Foliar fungicides containing quinone outside inhibitors(QoI), demethylation inhibitors(DMI), and succinate dehydrogenase inhibitors(SDHI) are commonly used to manage foliar diseases in corn. To mitigate the losses from tar spot thirteen foliar fungicides containing single or multiple modes of action (MOA/FRAC groups) were applied at their recommended rates in a single application at the standard tassel/silk growth stage timing to evaluate their efficacy against tar spot in a total of eight field trials in Illinois, Indiana, Michigan, and Wisconsin during 2019 and 2020. The single MOA fungicides included either a QoI or DMI. The dual MOA fungicides included a DMI with either a QoI or SDHI, and fungicides containing three MOAs included a QoI, DMI, and SDHI. Tar spot severity estimated as the percentage of leaf area covered by P. maydis stroma of the non-treated control at dent growth stage ranged from 1.6 to 23.3% on the ear leaf. Averaged across eight field trials all foliar fungicide treatments reduced tar spot severity, but only prothioconazole+trifloxystrobin, mefentrifluconazole+pyraclostrobin+fluxapyroxad, and mefentrifluconazole+pyraclostrobin significantly increased yield over the non-treated control. When comparing fungicide treatments by the number of MOAs foliar fungicide products that had two or three MOAs decreased tar spot severity over not treating and products with one MOA. The fungicide group that contained all three MOAs significantly increased yield over not treating with a fungicide or using a single MOA.
Xinhua Ding, Chongchong Lu, Mingxia Hao, Lingguang Kong, Lulu Wang, , Yurong Sui, Yingzhe Yue, , Ziyi Yin, et al.
Published: 12 January 2022
Abstract:
Rice (Oryza sativa L.) is the largest grain crop, accounting for about 40 % of the total grain production in China. In mid-July 2021, bacterial leaf streak-like disease emerged in rice varieties Chunyou584 and Yongyou2604 in Linyi city, Shandong Province, China. Disease incidences of the disease ranged from 80% to 90% in the surveyed fields. Infected rice leaves displayed dark green to yellowish-brown water-soaked thin streaks, and a large amount of beaded yellow oozes were observed on the lesions. After drying, there were gelatinous granules that were not easy to fall off and spread between leaf veins (Fig.S1A). According to the field symptoms of this disease, it was preliminarily suspected to be rice bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc), which is a guaranteed disease in China. To isolate the causal agent, leaf discs (~1 cm2) of diseased leaves were collected from the margins of the lesions, surface sterilized and ground into pieces in sterile double distilled water. The 10-3, 10-4 and 10-5 dilutions were spread onto peptone sugar agar (PSA) and incubated at 28°C for 36 hours. Yellow mucous bacterial colonies were consistently obtained on PSA medium. To identify the pathogen, fragments of the 16S rDNA, leuS and rpoB were amplified and sequenced using the primers previously reported (Yu et al. 2021). Three strains (LY01, LY02 and LY03) showed identical colony morphology and LY01 was used for further analyses. Sequence analyses showed that the fragments of 16S rDNA (955 bp, GenBank accession number: OK261898), leuS (755 bp, GenBank accession number: OK298387) and rpoB (926 bp, GenBank accession number: OK298388) of strain LY01 shared 99.16%, 99.46% and 100% similarities with those of Pantoea ananatis TZ39 (GenBank accession numbers: CP081342.1 for 16S rDNA, MW981338.1 for leuS and MW981344.1 for rpoB), respectively, which suggest the pathogenic bacterial strain LY01 isolated is P. ananatis. In addition, the single colony of P. ananatis LY01 was shown as Fig. S2B. Furthermore, pathogenicity tests were also performed according to the following steps. Bacterial suspension at OD600=0.1 was inoculated into eight rice leaves of four healthy rice plants (Chunyou 584) at 25-33°C and 60%-80% relative humidity in the field using a clipping method (Yang et al. 2020) or spraying methods, and sterile distilled water was as negative control. The clipped leaves (Fig. S1B) and spray-inoculated leaves (Fig. S1C) showed dark green water-soaked streaks at 14 days after inoculation, respectively, which showed similar symptoms with those samples collected from the fields (Fig. S1A). On contrary, the control rice leaves remained healthy and symptomless (Fig. S2A). The bacterium was re-isolated in the inoculated rice leaves and the re-isolated bacterial isolates, which was confirmed by sequencing 16S rDNA, leuS and rpoB, incited the same symptoms as in fields, which fulfills Koch’s postulates. In the past decade, P. ananatis was reported to result in grain discoloration and leaf blight in China (Yan et al. 2010; Xue et al. 2020, Yu et al. 2021), which could result in 40% - 60% yield losses. To our best knowledge, this is the first report of the bacterial leaf streak-likely disease occurred in Shandong Province caused by P. ananatis, so we named it as Pantoea leaf streak of rice. Although P. ananatis was also reported in Zhejiang province and Jiangxi province, which caused leaf streak lesions on rice, the disease symptoms are completely different from those of Pantoea leaf streak of rice. To the best of our knowledge, this is the first report of Pantoea leaf streak of rice caused by P. ananatis. This study provides sloid evidence that Pantoea leaf streak of rice in Eastern China can be caused by the new pathogen, P. ananatis, rather than Xoc as traditionally assumed. Disease development and quarantine of the new Pantoea leaf streak of rice disease caused by P. ananatis on rice need more attention in the near future.
Gensheng Zhang, Wei Liu, Xiangrui Cheng, Lin Wang, Xiaxia Tian, Zhimin Du, Zhensheng Kang,
Published: 12 January 2022
Abstract:
In 2017, a new race (TSA-6) of the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici, virulent to resistance gene Yr5 were detected in China. However, whether Chinese wheat cultivars are resistant to the new races was unknown. In this study, two isolates (TSA-6 and TSA-9) with virulence to Yr5 were tested on other wheat Yr gene lines for their avirulence/virulence patterns and used, together with prevalent races CYR32 and CYR34 without the Yr5 virulence, to evaluate 165 major Chinese wheat cultivars for their reactions. Isolates TSA-6 and TSA-9 had similar but different virulence spectra, and therefore should be considered as two different races. Their avirulent/virulence patterns were remarkably different from that of CYR34 but quite similar to that of CYR32. Of the 165 wheat cultivars, 21 had all-stage resistance to TSA-6, 34 to TSA-9, and 20 to both races. Adult-plant resistance (APR) was detected in 35 cultivars to TSA-6 and 27 to TSA-9, but only 3 cultivars showed APR to both new races. Slow rusting resistance was observed in 24 cultivars to TSA-6 and of 33 to TSA-9. Analysis of variance (ANOVA) of disease index indicated a significant difference among cultivars, but not among the four races. Based on the molecular marker data, a low percentage of wheat cultivars carried Yr5, Yr7, Yr10, Yr15, Yr26, and/or YrSP. As TSA-6 and TSA-9 can be a serious threat to wheat production in China, monitoring TSA-6, TSA-9, and other races are continually needed.
Peng Cao, Yuhui Fang, Zikui Zheng, Xia Han, Huixi Zou,
Published: 12 January 2022
Abstract:
Dendrobium officinale Kimura L., an endangered orchid plant, is a rare and precious Chinese herb and widely used to prepare Chinese traditional medicine (Zheng et al. 2005). In August 2021, significant indications of an unknown leaf spot disease were observed on greenhouse-grown D. officinale in Yueqing of Wenzhou (28.39°N, 121.04°E), Zhejiang Province, China, the main producing location of this orchid plant. Approximately twenty percent of plants surveyed showed typical infection symptoms. Initially, the symptoms appeared as small, circular black spots. As the disease developed, the center of the lesions was sunken with a black border. To determine the causal agent, 10 symptomatic plant samples were collected and all pieces from symptomatic plant leaves were used for isolating pathogen. Tissues between healthy and necrotic area were cut into pieces (5 × 5 mm, n=10), disinfected with 10% sodium hypochlorite for 1 minute, rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar medium (PDA) for 1 piece per plate, and incubated at 25℃ in a dark biochemical incubator. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. Twelve same fungal isolates were obtained from all symptomatic leave fragments, then DDO11 was chosen as a representative isolate for further study. The colonies showed white aerial mycelium after 5 days culture at 25°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 8-12 days culture. Conidia were spindle shape, five cells, four septa, average 29.3 × 8.5 μm (n = 30; length × width). The apical and basal cells were lighter in color, and most of them were hyaline. The middle three cells were darker in color, and mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 32.5 µm in average length, and the basal cell has a small appendage, 9.2 µm in average length, n=30. For fungal identification to species level, Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. The ITS, TUB2 and TEF-1α gene sequences of the representative isolate DDO11 were deposited in NCBI GenBank nucleotide database with accession numbers OK631881, OK655895 and OK655896, respectively. BLASTn analysis respectively showed 100%, 100% and 99.6% nucleotide sequence identity with Neopestalotiopsis clavispora strain accessions MG729690, MG740736 and MH423940, which indicated that the pathogen belonged N. clavispora. A maximum-likelihood phylogenetic analysis based on multi-locus sequence (ITS, TUB2, and TEF-1α) using MEGA X showed the similar result (Kumar et al. 2018). To verify pathogenicity, thirty 1-year-old healthy D. officinale plants of cultivar Yandang1 were used for inoculation tests. Spores of N. clavispora DDO11 were produced on PDA for 7 days at 28°C and washed with sterile distilled water, and the concentrations were adjusted to 1 × 106 spores/ml using a hemocytometer. Fifteen surface disinfected healthy plants were inoculated by spraying the suspension (2 ml, 1 × 106 spores/ml) and covered with plastic bags for 24 h, and another 15 plants treated with sterile distilled water were used as control. The plants were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber (Kiangnan, China) at 25°C with 12-h day/night cycle for 8 days (Cao et al. 2019). All inoculated leaves showed symptoms identical to those observed in the field. No disease occurred on the controls. The Neopestalotiopsis isolate was reisolated from the symptomatic leaves, and species identification was confirmed by the morphological and molecular method described above. N. clavispora has been reported to cause diseases on a variety of plants all over the world, such as strawberry (Gilardi et al. 2019), blue berry (Shi et al. 2021), Syzygium cumini (Banerjee et al. 2020), Macadamia (Qiu et al. 2020), and so on. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot on D. officinale in China. This report will help us to recognize the leaf spot disease of D. officinale and establish a foundation for future studies on N. clavispora to address effective management strategies.
, Wanxin Han, Zheng Li, Jianing Cheng, Yang Pan, Dongmei Zhao, Dai Zhang, Qian Li, Zhihui Yang,
Published: 10 January 2022
Abstract:
In July 2020, potato plants (cv. Xisen 6) showing characteristic symptoms of aerial stem rot were observed in a field in Fengning Manchu Autonomous County, Chengde, Hebei Province (northern China). The disease incidence in that field (5 ha in size) was more than 50%. Aerial stem rot of potato has increased in prevalence over recent years in Chengde, it can cause significant yield loss on susceptible cultivars such as Xisen 6 and Huangxin 226. Affected stem (light brown and water-soaked stem sections) pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were soaked in 200 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the crystal violet pectate (CVP) medium. The CVP plates were incubated at 28°C for 48 h. Colonies producing pits were restreaked and purified on Luria-Bertani (LB) agar plates. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R (Weisburg et al. 1991) and sequenced. Results of the Blastn analysis of the 16S rDNA amplicons (MZ348607, MZ348608) suggested that the isolates FN20211 and FN20222 belonged to the genus Pectobacterium. Housekeeping genes including acnA, gapA, icdA, mdh, proA and rpoS were also amplified using a set of primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ356250-MZ356261). To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between FN20211 and FN20222. The result of phylogenetic analysis showed that the isolates clustered with P. polaris type strain NIBIO1006T, which was isolated from potato (Dees et al. 2017). And the concatenated sequence of the six loci of isolate FN20211/FN20222 is 100% identical to those of the strains PZ1 (CP046377.1) and WBC1 (GCF_011378945.1), which were isolated from potato in South Korea and from Chinese cabbage in China, respectively. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20211 and FN20222 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline as control. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacteria-inoculated seedlings showed disease symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers as described above. Therefore, P. polaris isolates FN20211 and FN20222 fulfill Koch’s postulates for aerial stem rot of potato. P. polaris causing blackleg and soft rot on potato plants has been reported in European countries including Netherlands, Norway (Dees et al. 2017) and Poland (Waleron et al. 2019), and also in Pakistan (Sarfraz et al. 2019) and Russia (Voronina et al. 2021). To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in China. The stem rot poses a significant threat to the local potato industry, and further research on epidemiology and disease management options is needed.
Jinshao Li,
Published: 10 January 2022
Abstract:
Gastrodia elata, a traditional and important medicinal plant in China, it is used to numerous medical reasons. It is widely planted in Shaxi, Guizhou Province, China. G. elata grown in Guizhou is of high quality and an important source of income for the region. However, a root rot disease has been reported on G. elata in Guizhou in recent years, with an incidence rate of approximately 25%; this disease has markedly affected the plant growth and development. It causes what is referred to as a “rotten nest” and “empty nest”, significantly reducing the yield and medicinal value of G. elata. Eighty diseased G. elata samples were collected from August to December 2020 in Shaxi. Tissue dissection was used to isolate the pathogen on an ultra-clean workbench. In short, thew surface of G. elata was wiped with 75% alcohol for 30 s and then rinsed three to four times with sterile water. After the surface had dried, the skin from an infected area of the plant was cut into a net shape using a sterile scalpel. Eighty diseased tissue samples were placed on PDA (potato dextrose agar) medium using a sterile medical syringe needle and placed in an incubator at 25 °C for 7 days, and 61 fungal isolates with the same morphological characteristics were obtained from the diseased samples. Pure cultures of a putative fungal pathogen designated SX13 were obtained using the single-spore isolation and cultured on PDA medioum for identification and analysis. The colony grew in a circular shape, and the early hyphae were compact and white. A light-yellow ring appeared in the outer circle of the hyphae, and could be seen on both sides of the plate. The upper side of the colony turned white subsequently, and the lower side was light yellow. Identification of SX13 as Fusarium solani was primarily done based on morphological characteristics (Chitrampalam et al., 2018). Colonies produced macroconidia, which were sickle-shaped with two to five septa; most of them had three septa (length by width: 17.28 to 36.23 μm by 4.33 to 6.43 μm). Smaller conidia were fusiform, renal, or oblong, with no or one septum (length by width: 5.56 to 14.35 μm by 2.93 to 5.76 μm). Chlamydospore were also observed with diameters of ranging from 3.43 to 13.12 μm. Identification of SX13 was verified through DNA sequencing. Genomic DNA was extracted using the Biomiga Fungal gDNA Kit. The internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Schoch et al., 2012), β-tubulin (primers T1/T2) (O’Donnell and Cigelnik, 1997), and actin gene (ACT) region (primers ACT-512F/ACT-783R) (Carbone and Kohn, 1999) were PCR amplified, sequenced, and subjected to NCBI BLASTn homology matching analyses (GenBank Accession Nos. MW888340, MW892976 and MZ440809). High levels of sequence homology were observed with a F. solani reference sequence (Accession Nos. MT560378, ITS=100%; KU938955, β-tubulin=100%; KM231197, ACT=99%). To complete Koch's postulates, a conidial suspension (106 spores/mlcollected from isolate SX13 was inoculated onto nine G. elata root samples. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, plants were kept under high relative humidity in the dark at 25 °C for 7 days. Symptoms similar to the original outbreak were observed on all inoculated plants. In contrast, the negative control plants were healthy and unaffected. The SX13 was re-isolated successfully from the diseased tissues and verified based on morphology and sequencing as described above. To the best of our knowledge, this is the first report of F. solani causing root rot disease on G. elata in China. These findings provide a basis for further research on the management of this disease.
Published: 10 January 2022
Abstract:
Podocarpus macrophyllus (Thunb.) D. Don is used in many fields, including landscape, medicine, and forest interplanting. In July 2019, shoot blight was observed on P. macrophyllus at three nurseries in Harbin, China. Approximately 15% of plants had symptoms of the disease, which included rapid, synchronized death of leaves on individual branches. Eventually the whole plant wilted. Leaves and stems turned dark blue to brown. Ten infected vascular tissue samples from 10 individual plants were surface-disinfested in 0.5% NaOCl for 5 min, rinsed 3 times in sterile distilled water, and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 26°C. Six similar fungal isolates from ten samples were isolated and subcultured. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Colonies on PDA consisted of densely floccose aerial hyphae with light yellow and pinkish pigments. Microconidia were oval to obovoid or allantoid, 3.8 to 11.8 μm in length and 2.8 to 4.6 μm in width, mostly non-septate on carnation leaf agar (CLA). Macroconidia were naviculate-to-fusiform slender, 24.9 to 57.2 μm in length and 2.8 to 4.5 μm in width with 3- to 5- septate, with a beaked apical cell and a foot-shaped basal cell. According to these morphological characteristics, all isolates were identified as Fusarium spp. (Aoki et al. 2001 ). Genomic DNA was extracted from a representative isolate LHS1. The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha gene (TEF-1ɑ) and β-tubulin (TUB2) gene were amplified using the primers ITS1 and ITS4 (Yin et al. 2012),EF1-728F/EF1-986R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995), respectively. DNA sequences of LHS1 were deposited in GenBank (accession nos. MT914496 for ITS, MT920920 for TEF-1ɑ and MT920921 for TUB2, respectively). MegaBLAST analysis of the ITS, TEF-1a, and TUB2 sequences indicated 100%, 97.7% and 100% similarity with Fusarium concentricum isolate CBS 450.97 (accession no. MH862659.1 for ITS, MT010992.1 for TEF-1a, and MT011040.1 for TUB2, respectively). To determine pathogenicity, P. macrophyllus plants were grown in 10-cm pots containing a commercial potting mix (five plants/pot). At the 10 to 12 leaf stage, 10 healthy plants (2 pots) were inoculated by spraying 5 ml of a conidial suspension (4×106 spores/ml) onto every plant. Ten plants treated with sterile distilled water served as a control. The test was repeated twice. All plants were placed in a humidity chamber (>95% RH, 26℃) for 48 h after inoculation and then transferred to a greenhouse at 22/28°C (night/day). All inoculated wilted with leaves and stems turning dark blue to brown 15 days after inoculation. No symptoms were observed on the control plants. The fungus was re-isolated and confirmed to be F. concentricum according to morphological characteristics and molecular identification. To our knowledge, this is the first report of F. concentricum on P. macrophyllus in world. The disease caused a large number of plants to wilt and die, seriously impacting the ability of the horticulture industry to produce P. macrophyllus. Although this pathogen causes leaf and shoot blight symptoms, it is not clear if the pathogen is also a vascular wilt disease. The occurrence of the new disease caused by F. concentricum highlights the importance of developing management strategies to protect P. macrophyllus.
Ping Xie, Fei Teng Zhong,
Published: 10 January 2022
Abstract:
Rhapis humilis Blume is an ornamental plant for landscaping that is widely distributed in China. In February 2020, a leaf spot disease was observed on R. humilis in a nursery shed in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The disease incidence was more than 90%. The early symptom was small water-soaked lesions, which then turned into black necrotic spots. Eventually, the individual lesions coalesced into larger ones, leading to the death of diseased leaves. Ten diseased leaves were collected from the nursery. The diseased tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before pathogen isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28°C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RHPH-1, RHPH-2, and RHPH-3) were obtained. The colonies of the isolates were approximately 5 cm in diameter after 7 days. They were initially whitish and later became grayish white. The NaOH testing on MEA cultures was negative. No sporulation was detected after 30 days. The fertile structures of the specimens collected in the nursery were examined. Pycnidia were globose, measured 68 to 265 × 72 to 360 µm (n = 20), and mostly embedded. Conidia were aseptate, hyaline, and ellipsoid, measuring 3.6 to 6.5 × 2.2 to 2.7 µm (n = 30). Based on the morphological characteristics, the fungus was identified as in genus Phoma (Boerema et al. 2004). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), partial RNA polymerase II largest subunit (RPB2), and beta-tubulin (β-tub) loci of three isolates using primer pairs ITS4/ITS5, RPB2-6F/RPB2-7R, and BT2a/BT2b, respectively (Chen et al, 2015; White et al, 1990). The sequences were deposited in GenBank (ITS, MZ419364-MZ419366; RPB2, MZ562293-MZ562295; and β-tub, MZ562296-MZ562298). Based on BLAST analysis, the sequences of the ITS, RPB2, and β-tub all showed 100% similarity to Phoma herbarum Westend. (CBS 377.92, accession nos. KT389536 for ITS; KT389663 for RPB2; and KT389837 for β-tub). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of R. humilis were grown in pots, with one plant in each pot. The leaves were pinpricked with sterile needles before inoculation. They were inoculated with mycelial plugs of the isolates or sterile agar plugs (as control), with four plugs for each leaf. Five plants were used in each treatment. Disease symptoms similar to those in the nursery were observed on the inoculated plants 2 weeks after inoculation, whereas the control plants remained healthy. The fungus was reisolated from the symptomatic leaves and confirmed as P. herbarum by morphology and ITS analysis. P. herbarum was reported to cause leaf spot on Atractylodes lancea, Camellia sinensis, Elaeis guineensis, Lilium brownii, and Vetiveria zizanioides in China; Bituminaria bituminosa, Glycine max, Medicago sativa, and Pisum sativum in Australia; and Salvia nemorosa in Italy (Li et al. 2011; Li et al. 2012; Thangaraj et al. 2018). To our knowledge, the present study was the first to report P. herbarum causing leaf spot on R. humilis in China. P. herbarum seriously affects the supply of seedlings in R. humilis, and its epidemiology on R. humilis should be further studied.
Ning Qian, , Cailian Feng, , X. L. Lu,
Published: 10 January 2022
Abstract:
Orychophragmus violaceus, belonging to the Brassicaceae family, is widely grown in many provinces of China as an ornamental plant and also as a green manure crop. In December 2019, field investigations showed that a leaf spot disease occurred on O.violaceus with 50% to 80% incidence in Huize City, Yunnan Province of China. Infected leaves showed symptoms of small black point spots in the early stage of onset. The lesions are distributed throughout the leaves and finaly expand to 10-15 mm in diameter after 10-15 days of onset. At this time, the lesions are gray to black, and some have round patterns, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. The leaves with typical lesion symptoms were sampled and photographed, and then subjected to isolate and characterize the pathogen. Six pure cultures (HEYA2; HEYA4; HEYC6; HEYD7; HEYD8; HEYD10) were obtained by single-hyphae isolation. On PCA medium, colony can reach 27 mm after 7d, at 25°C in darkness. Aerial hypha is cottony with white to pale gray color, while the colony reverse is fawn to dark. on V8 medium, conidiophore solitary or clustered, erect or knee-curved, occasionally branched, pare brown, separated, 82–130 × 5–9 µm. Conidia are solitary, straight or slightly curved, inverted rod-shaped, pare brown to brown, with 6-10 transverse septa, 0-5 oblique and longitudinal septa, columnar beak, conidial bodies (47.7-)69.3-103.8(-119.6)(11.2-)16.6-23.6(-27.8) µm. Beak septum, pare brown, (29.2-)34.4-72.4(-101.3)(4.2-)6.6-9.5(-11.3) µm. Morphologically these isolates resembled species belonging to genus Alternaria (Simmons, 2007). Genomic DNA of each culture was quickly extracted from mycelia using QS method (Chi et al. 2009). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and translation elongation factor -1α (TEF -1α) genes were amplified according described procedures (White et al. 1990; Berbee et al. 1999; Liu et al. 1999;Sung et al. 2007; Carbone & Kohn 1999). The sequences obtained in this study were deposited in GenBank with accession numbers: MW867245, MW867246, MW867247, MW867248, MW867249, MW867250, MW882913, MW882914, MW882915, MW882916, MW882917, MW882918, MW882919, MW882920, MW882921, MW882922, MW882923, MW882924, MW882925, MW882926, MW882927, MW882928, MW882929, MW882930. Phylogenetic analysis was conducted with combined sequences of the four loci, using the maximum likelihood method and the maximum parsimony method. In the phylogenetic tree, the six isolates and Alternaria brassicae (CBS 116528) clustered together with high bootstrap support values (MLBS=100; MPBS= 100). Based on both morphological characters and phylogenetic results, the isolates were identified as Alternaria brassicae. Pathogenicity test of isolate HEYA2 was carried out on the detached leaves in a dark thermostat incubator at 25°C. Five pots per leaf were inoculated with mycelia plugs (5 mm in diameter), another five pots were inoculated with pure agar plugs and used as the negative control. In addition, conidia suspension (105 conidia/ml) of isolate HEYD8 were sprayed on 3-month-old healthy plants grown in a greenhouse at 22 °C–28 °C. The plants sprayed with sterilized water were used as negative controls. The test was conducted three times. After 5-7 days, the leaves inoculated with other the conidia suspension or the mycelium plugs showed brown necrotic lesions that are similar to the symptoms observed in the field, but the controls remained healthy. The pathogen was reisolated and confirmed to be A. brassicae, completing Koch’s postulates. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on Orychophragmus violaceus in China.
Published: 10 January 2022
Abstract:
Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.
Published: 9 January 2022
Abstract:
The production of wine grapes is gaining widespread popularity and being carried out on approximately 2,200 hectares of land in Japan. Scions grafted onto rootstocks generally have been imported from the EU, USA, New Zealand, and Australia into Japan. Unfortunately, viruses have spread in Japanese vineyards by slipping through the net of plant quarantine. Grapevine rupestris vein feathering virus (GRVFV), which was detected in a Greek grapevine accessions, is a member of genus Marafivirus in family Tymoviridae (El Beaino et al. 2001). GRVFV has been detected in many countries such as USA, Canada, Australia, New Zealand, Italy, Spain, Switzerland, Czech Republic, Uruguay, and Pakistan (Jo et al. 2015; Eichmeier et al. 2016; Xiao and Meng 2016; Blouin and MacDiarmid 2017; Reynard et al. 2017; Cho et al. 2018; Mahmood et al. 2019; Wu et al. 2020). Herein we report GRVFV infection in Vitis vinifera L. grapevines from Japan. In February 2021, dormant canes from 18 V. vinifera cv. Cabernet Sauvignon with leafroll-like disease symptoms, growing in a vineyard located in Kanagawa Prefecture, were collected. No typical vein banding symptom by GRVFV were observed in the grapevines during the growing season. Total RNA was isolated from the canes using an RNeasy Plant Mini Kit and QIAshredder (Qiagen, Valencia, CA), and subjected to cDNA synthesis using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). RT-PCR was performed with GRVFV_6156F and GRVFV_6600R primers for GRVFV detection (Reynard et al. 2017). The expected 445 nucleotides (nt) amplification product was obtained from four of 18 grapevines. Sequence analysis of the products revealed 91% identities to corresponding sequences of GRVFV isolates CHASS (KY513702) and Mauzac (KY513701) from Switzerland. Genome walking to determine the whole-genome sequence of the GRVFV isolates from the four grapevines was performed. Briefly, the upstream and downstream of the 445 nt amplification product were amplified from first-strand cDNA using gene-specific primers designed from the product and CHASS-specific primers. Each amplified fragment was Sanger sequenced. Next, gene-specific primers were designed to obtain the complete genome of GRVFV as 13 overlapping DNA fragments from each of the four grapevine samples. An identical complete genome of 6,704 bp was assembled from the overlapping DNA fragments using MEGA 10 software and named as NA1 isolate (DDBJ accession no. LC619667). Phylogenetic analysis of the NA1 genome and corresponding sequences of GRVFV from other countries showed that NA1 formed a cluster with isolate NZ ChTK0004 from New Zealand (MF000326; Supplementary Figure 1). In pairwise comparisons, the complete NA1 genome was most identical at 88% and 87%, respectively to isolates NZ ChTK0004 and Mauzac. The predicted amino acid sequences of NA1 polyprotein shared high homologies (96%) to the corresponding polyprotein sequences of NZ ChTK0004 and Mauzac, suggesting that NA1 is genetically similar to GRVFV isolates from New Zealand and Switzerland. The NA1-infected Cabernet Sauvignon was co-infected with Grapevine leafroll-associated virus 3, Grapevine virus A, and Grapevine rupestris stem pitting-associated virus according to RT-PCR assay for grapevine virus detection (Nakaune and Nakano 2006). The results underscore the importance of intensifying quarantine measures to prevent introduction of exotic viruses via contaminated wine grape vegetative cuttings.
Published: 9 January 2022
Abstract:
Hiroshima Prefecture has the highest production area of hydroponically grown Welsh onions (Allium fistulosum L.) in Japan. Since the cultivation began in 1988, root rot (Fig. 1A) followed by leaf browning (Fig. 1B) has caused significant economic losses. Approximately 80% (loss of 45 million JPY) of plant loss occurred from May to Sep 2009 (Shimizu, unpublished), and the disease was observed again in 2020. Diseased Welsh onions (five to six leaf stage) were collected in 2009. Abundant nonseptate hyphae of Pythium-like organisms were observed in the rotted roots (Fig. 1C). Disinfected symptomatic tissue samples were placed on NARF medium (Morita and Tojo 2007) and incubated at 25°C for 3 to 7 days. Six Pythium-like organisms were isolated, and their morphological features on a grass blade culture, potato carrot agar (PCA) (van der Plaats-Niterink 1981), cornmeal agar (CMA) and V8 juice agar (Miller 1955) were examined. Hyphal growth rates from 1–46°C were measured by culturing on PCA. The ribosomal internal transcribed spacer (ITS) regions and mitochondrial COI of the isolates were amplified and sequenced according to Ueta and Tojo (2016). All six isolates obtained showed similar morphology, hyphal growth rates, and sequences of ITS and COI. Detailed descriptions are provided here for the representative isolate 72 (MAFF246451). The isolate produced asexual structures but did not form sexual structures, including oogonia, antheridia, and oospores on all the media used. Hyphae were up to 6.8 μm wide. Appressoria were knob-like terminations (Fig. 1D). Sporangia were filamentous and indistinguishable from the hyphae. Zoospores (Fig. 1E) were formed at 5–25°C. The diameter of encysted zoospores ranged 7.4–10.1 (av. 8.9) μm (Fig. 1F). Cardinal temperatures for hyphal growth on PCA were 5°C min, 28–31°C opt, and 35°C max. The daily growth rate at 25°C was 15.0 mm per day. The sequence analysis of all isolates, including isolate 72 (GenBank ac nos AB700596 for ITS, LC630955 for COI) showed the present isolates belonged to Pythium Cluster B2a (Robideau et al. 2011) (Fig. 2). Based on these features, the six isolates were identified as Pythium Cluster B2a sp. In the inoculation test, isolate 72 was cultured on CMA at 25°C for 5 days. Mycelium disks (5 mm diam) obtained from the culture were placed on the primary roots of 8-day-old Welsh onion seedlings (cv Koutou), which were grown at a density of six plants on rock wool cubes moistened with tap water in a 50 mL plastic pot. The inoculated and non-inoculated plants were grown at 28°C for 7 days in a growth chamber. The experiment was repeated twice using three pots per replication. The plants inoculated with isolate 72 wilted, and their roots rotted 7 days after inoculation. No disease was found observed on the non-inoculated plants. The isolate of Pythium Cluster B2a sp. was consistently re-isolated from the diseased plants, thus, fulfilling Koch’s postulates. Pythium Cluster B2a sp. causing stem rot on lettuce has been recorded in Italy (Garibaldi et al. 2017). To our knowledge, this is the first report of Pythium Cluster B2a sp. on Welsh onions. Since significant losses to root rot of Welsh onion have occurred in Japan, identification of the causal organism will enable the development of management practices to reduces losses.
, , Z. X. Zhang, Zhiwen Feng, ,
Published: 9 January 2022
Abstract:
Potato (Solanum tuberosum L.) common scab can be caused by multiple pathogenic Streptomyces spp. worldwide. Potato tubers (cv. Favorita) with severe pitted common scab symptoms were observed at a small farm (2 hectares) during harvest in Anshun, Guizhou province in early May 2020. The disease incidence was around 10%, and symptomatic samples were collected to isolate the pathogen. Two isolates, ZR-IMU141 and ZR-IMU146 (Accession number MW995958 and MW995959 respectively), showed more than 99% sequence identity to S. stelliscabiei sequences (Accession No. HM018085). Five house-keeping genes for multi-locus sequence analyze (MLSA) of Streptomycetaceae were amplified, sequenced and uploaded to NCBI: atpD (MZ343164 and MZ343165), gyrB (MZ343162 and MZ343163), recA (MZ343166 and MZ343167), rpoB (MZ343168and MZ343169) and trpB (MZ343170 and MZ343171). All the genes show over 98% identity with S. stelliscabiei. Phylogenetic trees of 16S rRNA gene sequence and multi-locus sequence analysis (MLSA) were constructed. The two isolates contain pathogenicity genes txtAB, nec1, and tomA, which was confirmed by PCR. To complete Koch’s postulates, 9 potato seedlings (cv. Favorita, 15 centimeters high), were transferred to new pots and inoculated with spore suspensions of ZR-IMU141 and ZR-IMU146 (104 CFU/ml), or water as a negative control. Two months later, potato tubers inoculated with either ZR-IMU141 or ZR-IMU146 exhibited typical symptoms of potato common scab, such as superficial or deep, raised, pitted, or polygonal lesions like the field symptoms, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed identical to the original isolate by 16s rRNA gene sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Guizhou province, China. We believe that this report will draw attention to the study and management of the increased pool of scab pathogens in China.
, , Ruibian Zhao, Yuanhong Wang,
Published: 9 January 2022
Abstract:
Enterobacter cloacae is a symbiotic bacterium, which is one of the species in intestinal microbiota in many humans and animals. In some cases, it causes harmful diseases in humans. More and more studies showed that E. cloacae caused disease on plants, such as macadamia, ginger, mulberry, onion, chili pepper and rice. Garlic (Allium sativum L.) is one of crops with economic importance in the world. It is also widely grown in China. During 2018 to 2020, the naturally infected garlic bulbs from garlic fields in Kaifeng of Henan Province (34.55° N; 114.78° E) showed dry brown discoloration and rot symptoms. The diseased garlic seriously affected its edible value. Voucher specimens collected on June, 2019 were deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU190620. To identify the causal agent of this disease, the bulb tissues of infected garlic were surface-disinfested in 0.6% sodium hypochlorite, dipped in75% ethanol, and then dipped in sterile distilled water. These bulbs were plated on LB medium and incubated at 37℃. A number of white colonies grew on the medium after plating for 16 h. All colonies were round, white, opaque, smooth, and gram-negative, which is a typical characteristic of Enterobacter. To confirm the initial identification of the isolated bacterium, the fragments of 16S rRNA gene and gyrA gene of 6 colonies were amplified, respectively. The PCR products were purified and sequenced. All 16S rRNA and gyrA sequences were identical to each other. The sequences of 16S rRNA gene and gyrA gene were deposited in GenBank with accession numbers MW730711 and MW768876, respectively. BLAST searches were conducted using the sequences of 16S rRNA and gyrA. The results showed 99.72%, and 96.91% identity with the corresponding sequences of E. cloacae strain CBG15936 (CP046116.1), respectively. Phylogenetic trees were performed using the neighbor-joining (NJ) method of MAGA X based on the sequences of 16S rRNA gene and gyrA gene. Phylogenetic tree indicated that isolates are most likely E. cloacae. Pathogenicity tests were performed by puncturing garlic bulbs with a hypodermic needle, followed by dipping in bacterial suspension with the concentration of 2×108 CFU for 5 minutes. As control, the garlic bulbs were treated with sterile water. The inoculated and control were incubated at 30°C. 7 days after inoculation, brown discoloration and rot were developed on all inoculated garlic bulbs. No symptoms were observed in the control group.The symptoms were similar to that observed on the original diseased garlic bulbs. The garlic bulbs in inoculated and control were ten replicates in each independent biological experiments. The pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased garlics and confirmed as E. cloacae by morphological and sequence analyses as above. The re-isolated bacteria were identified by biochemical and physiological characteristics using API 20E strips. The results of the identification were identical to those of the edible ginger strains and the chili pepper strains. As far as we know, this is the first report of bulb decay on garlic caused by E. cloacae. The results are of great significance not only for the management of garlic bulbs during postharvest handling and storage, but also for the further research of opportunistic human pathogens E. cloacae.
, , , , Ruiting Li, Chen Liang, Xiaojuan Chen, Jianming Chen
Published: 9 January 2022
Abstract:
Bidens pilosa L., (spanish needle), is a wild, flowering plant of Asteraceae, that is grown in gardens, fields, roadsides, and riverbanks in Fuzhou, China. It is also used in traditional folk medicine for a broad range of ailments in China. In March 2019 and 2020, hundreds of B. pilosa growing along the roadsides, and gardens in the districts of Minhou and Jinshan were observed to be severely affected by a powdery mildew with approximately 80% disease incidence. Symptoms appeared as circular to irregular small white, powdery patches, typically on the adaxial sides of leaves and progressed to coalescent colonies on the leaves. As the disease developed, the infected leaves became wilted and senesced. Mycelia on leaves were superficial and solitary appressoria were slightly to distinctly nipple-shaped. Conidiophores were erect, 120 to 230 × 10 to 12 µm, and produced two to five conidia in chains with a sinuate outline. Foot-cells were erect, cylindrical, and 60 to 110 μm long. Conidia were hyaline, ellipsoid to barrel-shaped, 26 to 40 × 18 to 24 μm, and devoid of distinct fibrosin bodies. Germ tubes were long and produced at the perihilar position of the conidia. No chasmothecia were observed. Morphological characteristics overlapped with Golovinomyces ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012) on hosts within the Asteraceae tribe Heliantheae (Takamatsu et al. 2013). For molecular identification, ITS and IGS regions as well as partial LSU of two representative collections (MJU-IM019- MJU-IM020), were amplified using ITS1/ITS4, IGS-12a/ NS1R and LSU1/LSU2 primers (Carbone & Kohn, 1999; Scholin et al. 1994; White et al. 1990), respectively. The resulting sequences were deposited in GenBank (ITS: MW965777, MW965778; LSU: MW965787, MW965788; IGS: MW981256, MW981257). A BLAST search revealed 99 to 100 % sequence similarity to G. ambrosiae sequences (KX987303, AB769421, AB077689, AB769426, AB077643, and AB769425). Phylogenetic analysis of ITS, LSU and IGS also grouped obtained sequences within the G. ambrosiae complex (Qiu et al. 2020). Pathogenicity was confirmed through inoculation by gently pressing infected leaves onto leaves of five healthy, potted, young B. pilosa plants, while five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 ± 2°C. Inoculated plants developed symptoms after 7 to 10 days, whereas the control plants remained symptomless. The morphology of the resulting fungus on inoculated plants was identical to that originally observed on diseased plants. Podosphaera spp., have been reported on B. pilosa (Farr & Rossman 2021) from North America, Africa, and Asia. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on B. pilosa in China and Asia. Wild populations of B. pilosa may be the primary source of powdery mildew inoculum for commercial Asteraceae members and may warrant consideration in the control of this disease. References: Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, The Netherlands. Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 18 April 2021. Qiu, P. L., et al. 2020. BMC Microbiol. 20:1. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Takamatsu, S., et al. 2013. Mycologia 105:1135. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
, , You-Kyoung Han, , Jong-Han Park,
Published: 9 January 2022
Abstract:
Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.
Published: 9 January 2022
Abstract:
Japanese hop (Humulus scandens) is a non-native, invasive plant that colonizes disturbed riparian areas throughout the eastern United States and Canada, forming dense, monocultural stands that displace native plant communities due to a high reproductive rate, rapid growth, climbing bines, and dense shading (Balogh and Dancza 2008). It is capable of serving as a reservoir for agronomically important plant pathogens, such as the Tomato spotted wilt virus and powdery mildew species that infect commercial hemp and hop fields (Yoon et al. 2018; Weldon et al. 2020). In the spring of 2016, diseased populations of H. scandens were observed along the Monocacy River in Frederick County, Maryland with severe chlorotic and necrotic leaf lesions. Symptomatic leaves were surface sterilized and placed in moist chambers at 25°C for sporulation. Sporulating acervuli, lacking setae, developed on irregular, tan necrotic leaf lesions following 7 to 12 days in a moist chamber (Figure 1). Conidia were hyaline, aseptate, smooth-walled, fusiform to cylindrical with both ends acute (Figure 1B). Conidia measured (n = 100) [L x W; Average (+ Std. Err), range]: 12.42 µm (± 0.10), 8.41 – 14.48 µm; x 3.91 µm (±0.03), 3.03 – 4.91 µm. Monoconidial fungal cultures were obtained by transferring conidia with a sterile glass needle to acidified potato dextrose agar and incubated at 25°C for 2 to 3 days. Based on phenotypic characteristics and conidial morphology and size, the pathogen appeared to belong to the Colletotrichum acutatum complex (Damm et al. 2012). Therefore, six loci (ITS, GADPH, CHS1, HIS3, ACT, and TUB2) were amplified and sequenced from a representative isolate, 16-008, for species characterization (GenBank accessions MW023070 to MW023075) (Damm et al. 2012). For the ITS region and ACT, GADPH, and CHS1 loci, isolate 16-008 was 100% identical to C. fioriniae and shared 99% similarity to TUB2 and HIS3 for multiple accessions of C. fioriniae in GenBank. Gene sequences were aligned, trimmed, concatenated, and analyzed against 32 reference strains, within the C. acutatum complex (Damm et al. 2012). Concatenated loci were used to generate a maximum likelihood phylogeny using W-IQ-TREE (Trifinopoulos et al. 2016). Results from the phylogenetic analysis demonstrated that isolate 16-008 was most genetically similar to C. fioriniae with a bootstrap support of 100% (Figure 2). Based on phenotypic and sequence analyses, isolate 16-008 was identified as C. fioriniae. Humulus scandens seedlings from Maryland (n = 3) were inoculated with a conidia suspension (107 conidia mL-1) with 0.125% Tween 20® and applied with an atomizer until runoff. Inoculated plants were placed in a dew chamber at 25°C for 2 days. Experimental plants were distributed in a mist tent at 25°C with 14 h of light and monitored for 2 weeks. Negative control plants (n = 2) were sprayed with a sterile 0.125% Tween 20® water solution. All inoculated plants were symptomatic by 12 days post inoculation. No symptoms were observed on the mock-inoculated plants. Symptoms were identical to disease field samples. Inoculations were repeated with the same results. Colletotrichum fioriniae was reisolated and confirmed from excised leaf lesions via ITS and ACT sequencing. To our knowledge, this is the first report of C. fioriniae naturally infecting H. scandens within the United States (Farr and Rossman 2020). Future studies will evaluate the host range of this isolate due to the species broad host range and the weed’s extensive distribution.
Gabdiel E. Yulfo-Soto, Henry S Smith, , , ,
Published: 9 January 2022
Abstract:
In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar ‘White CBG’ being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars ‘Lifter’, ‘Trump Towers’, ‘Wife’ and ‘White CBG’ were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O’Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.
Yiwen Xu, Zhenyan Cao, Yihua Yang, , , , Xiaoping Yu
Published: 9 January 2022
Abstract:
Ophiopogon japonicus (Linn. f.) Ker-Gawl, a traditional Chinese medicinal plant, is widely cultured in China. The root of O. japonicus, is used as the main ingredient in many presriptions. It is rich in chemical components for steroidal saponins, homoisoflavonoids and polysaccharides, which have various pharmacological activities, such as cardiovascular protection, anti-inflammation and anti-diabetes (Chen. et al. 2016). In May and July for 2018 and 2019, the symptoms of black spot on O. japonicus were observed with an incidence of 40% in Cixi County, Zhejiang Province, China. The pathogen mainly infected leaves causing severe black spots, which resulted in a 28% yield loss per acre. At the early stage of the disease, the tip of the leaf began to turn yellow, then the discoloration gradually spread to the base of the leaf and finally the whole leaf turned reddish brown with visible black spot. Symptomatic leaves were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30s and 1% NaClO for 30s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA), and incubated at 28 ℃ in dark for a week. Then, pathogen on the PDA were transferred onto potato carrot agar (PCA), and incubated at 23 ℃ under the condition of alternation of day (12 h) and night (12 h) for a week. Colonies on PDA were dark gray in the center surrounded by white to gray on the upper side, and black with white margins on the back of the plate. Colonies on PCA were grayish with sparse hyphae. The conidia were obclavate or ellipsoid, pale brown, with 3~8 transverse septa and 1~4 longitudinal septa. Conidiophores were septate, arising singly, and measured (17.0~81.0) × (8.0~23.5) μm, Most conidia had a conical or columnar beak, approximately (0~23.5) × (2.5~9.0) μm in size. According to morphological and cultural characteristics, these isolates were preliminarily identified as Alternaria alternata. A. alternata is one of the most typical plant pathogen, more than 95% of which facultatively parasitize on plants, causing disease in numerous crops. To further confirm identification of pathogens, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF-1α), RNA polymerase Ⅱ second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), Histon 3 gene (His) and plasma membrane ATPase (ATP)were amplified with primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-7cr/RPB2-5f2, Alt-for/Alt-rev, His 3-F/His 3-R, ATP-F/ATP-R (Lawrence D.P. et al. 2013; Hong, S.G., et al. 2005). BLASTN analysis of NCBI using ITS (Accession NO. MW989987), Alt a1 (Accession NO. MW995953), EF-1α (Accession NO.MW995955), ATP (Accession NO.MW995957), His (Accession NO. MW995954) and RPB2 (Accession NO. MW995956) showed 100%, 100%, 97%, 99%, 99% and 97% identity to A. alternata MN249500.1, MN304714.1, MK637432.1, MK804115.1, MK460236.1, MK605888.1, respectively. To verify pathogenicity, healthy plants (1-year-old) of O. japonicus in ten pots were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70–80% relative humidity and a photoperiod of 16:8 [L: D] h). Fourteen days later, all inoculated plants showed typical symptoms of black spot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as A. alternata by morphology observation and sequence analysis. To our knowledge, this is the first report of black spot caused by A. alternata on O. japonicus in Zhejiang, China.
, , Shuo Zhang, X. Jin, Zhi Min Hao, Yunzhuan He
Published: 9 January 2022
Abstract:
Angelica dahurica (Fisch. ex Hoffm.) is an abundantly cultivated Chinese herbal medicine plant in China with about 4000 hectares grown, the annual production is up to 24,000 tons. The medicinal part of A. dahurica is its root, and mainly function for treat cold, headache, toothache, rhinitis, diabetes, etc. Besides, A. dahurica is also used as a spice in Asia. In September 2018, brown spot was observed on the leaves of A. dahurica in fields of Anguo City, Hebei Province, China. In the field investigated, the incidence of brown spot disease reached 15%. The infected leaves showed brown spots surrounded with pale yellow edge, resulting in withered of the whole leaf. It seriously endangers the growth of A. dahurica, reducing the yield and quality of medicinal materials, even leading to the death of plants. We isolated the pathogen from 10 leaves with same lesions, the small square leaf pieces of approximately 3 to 5 mm were obtained with the sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min followed by washing in sterile water for 3 times, then incubated on potato dextrose agar (PDA) plates at 25°C for 4 days. The culture was transferred to new PDA plates and was cultivated in dark at 25°C for 10 days. A total of 3 species of fungi were isolated, and only one fungus species has been found to be able to cause the original pathological characteristics of A. dahurica leaves through the back-grafting experiment. The mycelium was black and began to sporulate after 8 days on PDA media by single spore separation. Multiple spores joined together to form spores chain. The spores were spindle-shaped, yellow to yellow brown, and size ranged from 45 to 55 × 15 to 20 µm (n=50), with zero to three longitudinal septa and one to five transverse septa. For pathogenicity tests, the spore suspension (3.5×105 spores/mL) were inoculated to healthy plants grown in experimental field, the test was repeated four times, and 10 leaves were inoculated in each repetition, and the sterile water was inoculated as the blank control. Inoculated leaves were covered with transparent plastic bags for 24 h to keep humidity. Nine days later, it was found that there were lesions on the leaves inoculated with the pathogen, and the traits were the same as those in the field, while the controls are healthy. The fungus was consistently isolated from the inoculated leaves. The similar isolates were re-isolated from the inoculated and infected leaves and identified as Alternaria tenuissima by DNA sequencing, fulfilling Koch’s postulates. Fungal genomic DNA was extracted from 7-day-old culture. PCR amplifications were performed using primers ITS1 / ITS4 and TEFF / TEFR respectively (Takahashi et al. 2006, Du 2008). The nucleotide sequence of PCR products, which have been deposited in Genebank under the accession numbers MN153514 and MN735428, showed 99.8%-100% identity with the corresponding sequences of A. tenuissima (MW194297 and MK415954). In order to further identify the pathogen species, we constructed a phylogenetic tree by combining TEF sequence and ITS sequence to distinguish the relationship between the pathogen and other minor species in the genus Alternaria, the isolate was clustered in the Alternaria clade. Therefore, the pathogen was identified as A. tenuissima based on the morphological characteristics and molecular identification. To our knowledge, this is the first report of A. tenuissima causing leaf spot on A. dahurica in China.
Jingya Zhao, Mengya Peng, , Xiaoping Xing, Yixuan Shan, Zhuo Fan, , , Xue Yang, , et al.
Published: 9 January 2022
Abstract:
Fusarium pseudograminearum is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Fusarium crown rot and Fusarium head blight in wheat. The basic leucine zipper proteins (bZIPs) are evolutionarily conserved transcription factors that play crucial roles in a range of growth and developmental processes and the responses to biotic and abiotic stresses. However, the roles of bZIP transcription factors remains unknown in F. pseudograminearum. In this study, a bZIP transcription factor Fpkapc was identified to localize to the nucleus in F. pseudograminearum. A mutant strain (Δfpkapc) was constructed to determine the role of Fpkapc in growth and pathogenicity of F. pseudograminearum. Transcriptomic analyses revealed that many genes involved in basic metabolism and oxidation-reduction processes were down-regulated, whereas many genes involved in metal iron binding were up-regulated in the Δfpkapc strain, compared with the wild type. Correspondingly, the mutant had severe growth defects and displayed abnormal hyphal tips. Conidiation in the Fpkapc mutant was reduced, with more conidia in smaller size and fewer septa than in the wild type. Also, relative to WT, the Δfpkapc strain showed greater replaced by increased tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but the significantly increased TRI genes expression, compared with the wild type. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.
Published: 9 January 2022
Abstract:
Upon reintroduction of hemp in 2014 and legalization in 2018, labeled pesticides have remained limited. Further, consumer demand has aimed the market toward organic or chemical-free production systems. In efforts to manage diseases and pests in fields and greenhouses, producers turn toward biological and biorational formulations. Efficacy of these fungicides against common aerial diseases of hemp is largely unpublished. Challenges of efficacy testing, however, further delay or discourage research. In this study, we evaluated screening methods against some common biological products. The aim was to test a screening model in order to examine products against fungal pathogens and to identify demonstrable differences under controlled conditions. Thus, in this study, we prescreen 11 biological and biorational fungicides against four common fungal leaf and flower pathogens using three bioassays. Confirmation that the major modes of action for these products have measurable activity against major pathogens of hemp serves as a first step toward more complex field studies.
Huan-Yu Chen, Chun-Chi Lin, ,
Published: 9 January 2022
Abstract:
Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch’s postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.
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