Biological And Molecular Characterization Of The Rare Orange-Sclerotial Isolates Of Botrytis Cinerea

Botrytis cinerea is a worldwide plant pathogenic fungus,causing gray mold disease in many plants and bringing a serious economic loss.It usually produces grayish to brownish mycelia/conidia and black sclerotia due to accumulation of1,8-dihydroxynaphthalene（DHN）melanin.Here,we report six rare isolates of B.cinerea stably producing orange sclerotia,and named them as orange-sclerotial（or OS）isolates.The isolates stably producing black sclerotia were named as black-sclerotial（or BS）isolates.In this study,biological and molecular characteristics of the OS isolates of B.cinerea were studied.Main results achieved were summarized below:（1）The taxonomic status of the OS isolates was clarifiedMorphological and molecular identification showed that the OS isolates belong to B.cinerea.The orange-sclerotial formation trait in the OS isolate XN-1 was found to be stably inherited through asexual reproduction.Results also showed that the OS isolates did not greatly differ from the BS isolates in mycelial growth,sclerotial formation,conidial production and germination,pathogenicity.The OS sclerotia germinated very rapidly upon maturation at 20°C to produce conidiophores and conidia without visible dormancy period.On the contrary,the BS sclerotia did not germinate directly upon maturation at 20°C.They usually need low-temperature conditioning before germination.（2）Low incidence of the OS isolates was observed under the natural conditionsA large field survey in two successive seasons（2012 and 2013）on natural occurrence of the OS isolates was conducted in 15 counties（cities）in Hubei Province.Results showed that five out of 1580 isolates of B.cinerea（accounting for 0.32%）from tomato and strawberry produced OS sclerotia and they are the OS isolates.Meanwhile,a fixed point survey in Xian‘an County,Hubei province（the place where the first OS isolate XN-1 was found）was done in two seasons（2012-2013,2013-2014）.Results showed that none of the 451 isolates of B.cinerea from strawberry,lettuce and leek produced OS sclerotia.（3）Ecological fitness of mycelia,conidia and sclerotia for the representative OS and BS isolates was elucidatedThe OS isolate XN-1 and the BS isolate B05.10 were compared for mycelial growth,production and germination of conidia,production,germination and survival of sclerotia,response of mycelia,conidia and sclerotia to abiotic and/or biotic stresses,and pathogenicity on oilseed rape and tobacco.Results showed that XN-1 did not differ or differ slightly from B05.10 in all the above-mentioned parameters except the sclerotia-related parameters.Compared to the BS sclerotia,the OS sclerotia survived poorly at 10°C,20°C and 30°C,and in response to abiotic stresses（high osmotic conditions,SDS,H₂O₂）,and the mycoparasitism by Trichoderma koningiopsis T-51and Clonostachys rosea S-48.These results suggest that low ecological fitness for the OS sclerotia might be responsible for low frequency of the OS isolates under natural conditions.（4）A single-nucleotide deletion(ΔG ^{nt 1524})was found in the transcription factor gene Bcsmr1 and it was proposed to be responsible for formation of OS sclerotiaSix sclerotial DHN-melanin biosynthesis-related genes,including Bcpks12,Bcpks13,Bcygh1,Bcbrn1,Bcbrn2 and Bcscd1,and three transcription factor genes,including Bcsmr1,Bcztf1 and Bcztf2 in representative OS and BS isolates were PCR cloned and sequenced.At the same time,expression of these genes were analyzed using q RT-PCR.The results indicated that a single nucleotide-deletion（SND）at nt1524 within the ORF of Bcsmr1(named asΔG ^{nt 1524})was observed in the OS isolates.The SND-mediated mutation caused truncation of the polypeptide Bc Smr1 and the shortened Bc Smr1 was lower than the full-length Bc Smr1 in activation of transcription of melanogenic genes,including Bcpks12,Bcygh1,Bcbrn1/2 and Bcscd1in OS sclerotia.Introduction of Bcsmr1 from the BS isolate B05.10 into the OS isolate XN-1 enhanced expression of Bcpks12,Bcygh1 and Bcbrn1,thus improving melanogenesis in the OS sclerotia of XN-1.（5）Transcriptomic profiling further confirmed that formation of OS sclerotia was caused by deficiency in expression of DHN-melanin biosynthesis genesWe found that expression of Bcpks12,Bcygh1and Bcbrn1 was down regulated in OS sclerotia OS sclerotia,compared to that in BS sclerotia.We also found that five genes（2 carbohydrate activation enzyme genes,3 genes for oxidation-reduction enzymes）in OS isolate had the similar expression patterns as Bcpks12,Bcygh1and Bcbrn1.The result of GO enrichment analysis showed that a lot of oxidation reduction enzyme genes enriched in the immature sclerotia of XN-1,indicating that the intracellular oxidation-reduction imbalance might occur in the OS sclerotia.In addition,the enrichment analysis of the common differentially expressed genes at different sclerotial development stages of B05.10 and XN-1 was conducted.The results indicated that biosythesis of antibiotics,fatty acid biosythesis and metabolism,transporter and biosynthesis of the secondary metabolites were found to play an important role in sclerotial development.Through the KEGG pathway enrichment analysis,we found that methane metabolism was closely related to sclerotial development.Finally,we isolated 303 candidate genes including 6 genes in a gene cluster,7 secondary metabolites biosynthesis related genes and 17 transcription factor genes were related to sclerotial developmen base on differentially expressed genes screening and the cluster analysis.The results advanced our knowledge about melanogenesis pathway and about sclerotial development in B.cinerea.The information provided a good example for understanding of the importance of sclerotia and melanin in life cycle of B.cinerea.