The Population Structre And Genetic Analysis Of Lophodermium On Pine Needles In Sichuan

Pine tree is one of the most important forestry species in the Northern Hemisphere.In China,there are 38 species and 12 variaties of pine tree including introduced cultivars,and their distribution is all over the entire country.Sichuan has the highest pine tree species among all of other provinces,main species are Pinus.yunnanensis,P massoniana,P. armandii,P.tabulaeformis,P.densata,P.elliottii,P.bungeanae,and P.kesiya var. langbianensis,etc.Sichuan forestry region is an important part of The Tibetan Plateau forestry, and its vegetation is the essential natural barrier to balance the ecosystem of the Yangtze River watershed area.The pine needle-shedding is a common disease throughout the world,which harms all seedlings,nurseries and mature forests.This disease can cause different levels of damage to pine trees from partially needles shed and fall to loosing up to 70%of the total needles.The loosing of the needles can severely damage the growth of the pine tree,decrease the timber volume to up to 80%,and even result in the death of the entire pine tree.Due to the wide-range affecting areas and the severe consequences,attentions have been paid largely on this disease.The pathogenic fungi which cause the pine needle-shedding disease belong to the genus Lophodermiun within the fungi family Rhytismataceae.More than 100 varieties have been reported in this species on both conifers and broadleaves.Due to its wide ranges of distribution and host plants,researches have been focusing on the variety identification and its development systems.This research based on multiple years investigation on the pine needle-shedding disease in Sichuan,and emphasized on identification of the virulent varieties, genetic diversity,and their systematic development relationships.The results from this research can provide the scientific basis to prevent the pine needle-shedding disease and maintain the health of Sichuan forestry region.1.Based on the morphological classifications of Lophodermium on pine by Minter (1981),7 Lophodermium fungi from samples collected more than 20 geographic locations in Sichuan are identified.They are Lophodermium conigenum,Lophodermi pinastri, Lophodermium sichuanense,Lophodermium indianum,Lophodermium austral, Lophodermium erlangshanense(new species going to register),and Lophodermium kumaunicum.Based on the morphological traits,a classification searching index of Lophodermium on pine in Sichuan was also developed:2.By the identification of the existing ascocarp on pine needle from 400 randomly selected pine trees in 29 different forestry regions in Sichuan province,the pine tree needle-shedding disease were found from all of the locations surveyed in this research,and the infection levels could be as high as 50%.The pathogen Lophodermium conigenum and Lophodermi pinastri existed on five pine tree cultivars P.yunnanensis,P massoniana,P. elliottii,P.densata,and P.armandii,and frequently growing together.Pathogen Lophodermium indianum existed on the needles collected from the ground of pine tree P.yunnanensis,P.elliottii,and P massoniana.Pathogen Lophodermium australe was only found growing on the needles of pine tree P.elliottii,and pathogen Lophodermium kumaunicum only appeared on pine tree P.densata as scarce events.It was also discovered that pathogen Lophodermium sichuanense only appeared on living needles of P.yunnanensis in Mount Erlang.Pathogen Lophodermium erlangshanense were found on both living or fallen needles of P.armandii with small amount on Mount Erlang and Mount Paoma in Kangding.3.Ascocarp from fungi Lophodermium collected from various geographic locations in Sichuan were separated and cultivated.Totally 24 strains(No.from L-01 to L-24) were obtained and their morphological traits were described based on each colony.Various cultivation conditions were analyzed including nutrient concentrations,carbon sources and types of culture media,and the germination and growing of Lophodermium were assessed on PDA plates.No sexual organs were found after 60 days cultivation at 23℃.4.Total DNA was isolated from the cultivated Lophodermium by two methods adapted from twice deposition and benzyl chloride extraction for RAPD analysis and ITS sequencing. Both of the methods proved to be effective in isolating DNA for this purpose.The essential steps of successfully extraction DNA were before DNA extraction,freeze the mycelium in-20℃for 12h in refrigerator,then homogenize the frozen mycelium in mortar,and then add corresponding extracting solutions.5.After cycles of screening,11 primer pairs were selected from initial 160 primer pairs for RAPD analysis of 24 Lophodermium strains.The PCR reaction conditions were also optimized and the best conditions were:25μl PCR reaction volume,10×Taq enzyme buffer (2.5μl),15UTaq enzyme,20ng DNA template,0.4 mmol/L dNTPs,4.0 mmol/L Mg²⁺, 0.48μmol/L primers,and 10.2μl ddH₂O.6.The results from the RAPD analysis of 24 strains of Lophodermium by using 11 primer pairs found 217 sites were amplified.Among the 217 bands,214 sites were indentified as polymorphisms,which accounted for 98.6%of total amplified sites,and evidenced the abundant genetic diversity of Lophodermium in Sichuan.The clustering chart of the 24 strains of Lophodermium was developed based on the coefficients of comparability by using UPGMA.It demonstrated that when comparability coefficient is 0.28,all the testing strains could be divided intoâ… ,â…¡,â…¢,â…£,andâ…¤types,and typeâ… could be further divided into subtypeâ… -1 andâ… -2,as subtypeâ… -1 contains L.pinastri and L.conigenum which shares multiple morphologic traits.Typeâ…£andâ…¤separated from others due to their low affinity, and on the other hand evidenced the relationship between biology and morphology.Analysis on the fungi strains of L.conigenum which only infect the same host P massonictna found a broad genetic diversity within the same genus,and it was mainly due to the geographic differences.On the other hand,genetic diversity within same genus could also be results of different host origins,which was proved by the matrix analysis of the comparability coefficient for fungi strains from both L.pinastri and L.conigenum collected from Mount Erlang in Luding.7.Analysis of base sequencing in ITS region of rDNA of 18 strains of pine Lophodermium in Sichuan was conducted by using fungi universal primer ITS₃ and ITS₄,and the result has registered in GenBank(AY422489,AY422490,AY515318,AY515319, EU696766,EU696767,EU696768,EU696769,EU696770,EU696771,EU696772, EU696773,EU696774,EU696775,EU696776,EU696777,EU696778,EU696779). Multi-sequence analysis of ITS region was conducted by using DNAstar,and the systematic development tree was constructed.Similar to the RAPD results,18 strains could be divided into 5 groups.Compared with 9 Lophodermium's ITS region registered in GenBank from Mexico,USA,and Finland,the homologous rate reached up to 92.9%.The homologous rate between Lophodermium sichuanense and Lophodermium from European is low and only ranged from 70%to 80%.Above all,due to the complex geological environment in Sichuan forest and the diverse species of pine,the lophodermium on pine needle contained 7 genuses,and 2 of them are new species reported from Sichuan.Genetic diversity was largely observed within or between species.The classifications based on results from morphological traits,RAPD,and base sequencing analyses in ITS region of rDNA are consistent.How to utilize the modern technology of molecular marker to identify the pathogen of pine needle cast,to diagnosis in early stage,and to provide scientific basis of disease prevention and control are the future emphases that need to be studied further.