First Report of Species of Colletotrichum Causing Leaf Spot of Liriodendron chinense × tulipifera in China

Abstract

Hybrid tulip tree (Liriodendron chinense (Hemsl.) Sarg. × tulipifera L.) is an important landscaping tree widely planted in China. Leaf spot symptoms on hybrid tulip trees were observed on the campus of Nanjing Forestry University (NJFU), China from May to Nov. 2017. The spots were initially 1–2 mm diam. with yellow edges, and then enlarged and turned brown or black. The lesions coalesced and became elongated or irregularly shaped, and rarely acervuli and pink conidial masses developed. Later, the lesions became necrotic and shriveled. Small pieces (3-4 mm2) from the margin of an infected lesion were surface-sterilized in 75% ethanol for 30 s, 1% NaOCl for 90 s, and were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and 2 isolates (G2 and R3) were obtained and deposited in China’s Forestry Culture Collection Center (G2: cfcc53053, R3: cfcc53054). When isolate G2 was cultured on PDA, the colony was grayish-white, and pale orange towards the center on the reverse side. The conidia were 1-celled, straight, hyaline, subcylindrical with rounded ends, and 15.3±1.1×6.3±0.3 µm in size. Appressoria were one-celled, pale brown, thick-walled, ellipsoidal, and measured 8.4±1.1×6.4±0.7 µm. When isolate R3 was cultured on PDA, the colony was white and then turned grey and light grey on the reverse side, producing dark–green pigmentation near the center. The conidia and appressoria were almost identical with G2, with sizes of 15.0±0.9×4.8±0.1 µm and 7.7±1.1×6.5±0.6 µm, respectively. The characteristics of the conidia and other structures of G2 and R3 matched the morphological characteristics of the C. gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS) or actin (ACT) were respectively amplified with primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, or ACT-512F/ACT-783R. The sequences were deposited in GenBank [Accession Nos. MK268673 to MK268676 for G2, MK268677 to MK268680 for R3]. A Blast search of GenBank showed that ITS, ACT, CHS and GAPDH sequences of G2 were identical to C. gloeosporioides at a high level (>99%). For R3, the ITS was 100% identical to C. gloeosporioides CBS 125969 or C. siamense ICK-3, the GAPDH and ACT were 99% identical to C. siamense ICK-23 and COUFAL0199, respectively, while the CHS was 98% identical to C. fructicola ICKA 15. Phylogenetic analysis using neighbor joining and concatenated sequences (ITS, GAPDH, CHS, and ACT) with MEGA 7 placed G2 in the clade of C. gloeosporioides s.s. with extype ICMP 17821 and R3 in the clade of C. siamense with extype ICMP 18578. Based on these morphological and phylogenetic studies, G2 and R3 were C. gloeosporioides s.s. and C. siamense, respectively. Pathogenicity of the two species was verified on detached and attached leaves inoculated with 10 μL of conidial suspension (106 conidia/mL) and 5-mm plugs cut from the edge of 5-days old colonies, each with 5 replicates. Controls were treated with sterile dH2O and agar plugs. All detached leaves were placed in 20-cm dishes on wet filter paper at 25°C. Each attached leaf and a wet cotton ball were covered with a plastic bag on the trees for 24 h. Spots appeared in 2 days and reached 2 cm diam. in 5-8 days. The symptoms were the same as those in the field, but with no yellow edges. Recovery rates of G2 and R3 were 90% and 80%, respectively. No lesions were observed on control plants. C. gloeosporioides and C. acutatum were previously reported infecting L. tulipifera (Lori et al, 2004; Choi et al. 2012), but this is the first report of C. gloeosporioides s.s. causing tulip tree leaf spot in China and the first report of tulip tree as a host of C. siamense. All tulip trees at NJFU showed infection, and the level of disease was severe throughout Nanjing. Determination of the pathogens, C. gloeosporioides s.s. and C. siamense laid the groundwork for future studies of this disease.