Species Of Citrus Anthracnose Pathogens And The Population Genetic Diversity Of Dominant Species Colletotrichum Gloeosporioides In China

Anthracnose, caused by the genus of Colletotrichum, is one of the most important diseases of citrus all over the world. The pathogens infect young shoot, leaf, flower, petiole, young fruit and mature fruit of citrus, result in shoot and leaf blight, leaf spot, necrosis of flower and subsequent postbloom fruit dropping (PFD), premature fruit dropping and fruit decay postharvest. Huge losses had been resulted in historically as anthracnose disease was prevalent. However, the research of citrus anthracnose is worldwide limited, and this situation is more significant in China, the largest citrus-producing country in the world. In this paper, we studied the species of citrus anthracnose pathogens and the population genetic diversity of dominant species Colletotrichum gloeosporioides. The results were reported here.Two hundreds and twenty single-spore isolates of Colletotrichum spp. were collected from infected citrus organs, including leaf, shoot, twig, petiole and young, as well as mature fruit with different varieties from Zhejiang, Jiangxi, Guangdong, Fujian, Hunan, Chongqing and Yunnan provinces. They were identified to be three types based on culture characteristics and morphology of conidia. Type A:the colony is variable, white to dark blue in PDA, fast-growing with the rate of 1.2 to 1.7 cm/day; produced bright red spore masses, conidia with rounded ends, 12.5-18.6×4.7-6.0μm; B:gray colony, slow growing with rate of 0.6-0.8 cm/day, produced light red spore masses, conidia with pointed ends,4.1-5.1×12.4～14.8μm; C:cyan colony, growth rate:1.0-1.3 cm/day; produced orange spore masses, crescent-shaped conidia.Pathogenicity test demonstrated that both Type A and Type C were virulent to shoot, flower and mature fruit of Pankan(Citrus reticulata), induced typically necrotic symptom, whereas Type B is only virulent to the flower of Pankan. PCR amplification showed that the expected fragment of about 450 bp was only obtained from the isolates of Type A with C. gloeosporioides specific primers CgInt/ITS4, while the expected fragment of about 490 bp was only obtained from the isolates of Type B with C. acutatum specific primers CaInt/ITS4. There was no any fragment could be amplified with these primers for isolates of Type C. The ITS sequences were amplified with universal primers ITS4/ITS5 from selected isolates of these three types and sequenced. Sequence alignment showed that the ITS sequences from Type A (HN-L01, JX-F10, CQ-L03, GD-B15) were 99% identity to that of C. gloeosporioides (AJ301988 and AM947679). The ITS sequences from Type B(YN01, YN02, YN-3-16) were 99% identity to that of C. acutatum (AJ301964 and AJ301932). The ITS sequences from Type C(XY-01, XY-02) were 99% identity to that of C. truncatum (AY266380 and AJ301944). Thus, Type A and B were identified as C. gloeosporioides and C. acutatum, respectively. Type C was temporary identified to C. truncatum. This is the first report that C. truncatum is present and pathogenicity to citrus, but further work is necessary for final conclusion.Among the colleted isolates, C. gloeosporioides was the dominant species, with the ratio of 96.3%, present in all provinces investigated. C. acutatum was only found on key lime (C. aurantifolia) in Yunnan province, with the ratio of 1.4%; C. truncatum was collected from Yunnan and Guangdong, with the ratio of 2.3%.To analyse the population genetic diversity of Colletotrichum gloeosporioides, the dominant species of anthracnose fungi of citrus in China, partial sequences of five genes including beta-tubulin-2-gene (BT), glutamine-synthetase (GS), complete ITS, glyceraldehyde-3-phosphate dehydrogenase (GDPH) and calmoudulin (Cal) were amplified using primers Bt2a/Bt2b, GSF1/GSR1, ITS4/1TS5, GDF1/GDR1, CL1/CL2, respectively. Phylogenetic trees were constructed by method of neighbor-joining (MEGA4.0). The results showed that all 37 citrus isolates of C. gloeosporioides clustered into one same group if using ITS or BT sequences to construct, suggesting that either ITS or BT could not distinguish the intraspecific variation of C. gloeosporioides. However, phylogenetic analysis with the sequences of Cal, GDPH and GS genes revealed that these isolates could be grouped to Cal-Ⅰand Cal-Ⅱ, GD-Ⅰand GD-Ⅱ, and GS-Ⅰ, GS-Ⅱ, GS-Ⅲ, GS-Ⅳ, respectively, suggesting that the these genes were effective to distinguish the intraspecific variation of C. gloeosporioides. A high level of genetic differentiation among C. gloeosporioides from citrus was detected by phylogenetic analysis using the combined sequences these 5 genes, They were grouped into 2 groups, Group A included subgroupsⅠto subgroupⅥ, and Group B included subgroupⅧand subgroupⅧ.The genetic differentiation of C. gloeosporioides isolates from citrus was not associated with citrus varieties, host organs, geographical origin, but was associated with shape and size of conidia. GroupA has longer and narrower conidia, 16.3～18.6×4.7-5.3μm, arverged 16.9×5.0μm,Group B has shorter and wider conidia,12.5～14.7×4.9-6.0μm, averaged 13.9×5.4μm.