Colletotrichum Species Associated With Anthracnose Of Pyrus Spp. In China And Interactions Between Colletotrichum Fructicola And Host Pear

China is the greatest pear production country in the world,and its cultivated area and total yield of pear fruits both rank the first.However,in recent years,the occurrence of pear anthracnose in major pear-cultivation areas of China has been significantly aggravated,resulting in a large number of fruit rot on the main varieties such as ‘Dangshansu' Pear and early defoliation of sand pear in southern China,which has posed a new serious threat to the development of the pear industry.To clarify the species composition,distribution and damage characteristics,as well as the morphological,genetic and pathogenic diversity of the pathogen Colletotrichum spp.from different regions and the Pyrus spp.in China,this study has conducted the identification and diversity analyses of Colletotrichum spp.collected from different pear-cultivation provinces,Pyrus species and varieties in China.In addition,to determine the pathogenic mechanism of C.fructicola at different stages of infection and the respective defense strategy of host pear leaves at the transcriptional level,the simultaneous transcriptome analysis of C.fructicola and pear leaf pathosystem were systematically carried out.The obtained results provided the first systematic investigation of this disease,and determined the morphological,molecular,biological characterization and diversity of Colletotrichum spp.associated with Pyrus plants in respect to the organ type and geographical location in China.This study contributed to provide useful theoretical basis for effective disease diagnosis and management,and useful information to help understand the pathogenic mechanism of the Colletotrichum spp.involved in pear anthracnose and disease resistance mechanism of the host plants.The main results were as follows:1.Identification and genetic diversity of Colletotrichum species associated with anthracnose of Pyrus spp.in China.A total of 297 samples were collected from cultivated pear species(including P.pyrifolia,P.bretschneideri,and P.communis)from seven major pear cultivation provinces in China during 2014 to 2016.The pear leaves and fruits affected by anthracnose were sampled and subjected to fungus isolation,resulting in a total of 488 Colletotrichum isolates.Totally,90 representative isolates were chosen for further analyses based on their morphology(colony shape,color,and conidial morphology),ITS sequence data,symptom type,origin,and host species involved.Phylogenetic analyses based on six loci(ACT,TUB2,CAL,CHS-1,GAPDH,and ITS)coupled with morphology of 90 representative isolates revealed that they belong to 12 Colletotrichum species,including four well-characterized species(C.aenigma,C.fioriniae,C.fructicola and C.gloeosporioides),six new record species(C.citricola,C.conoides,C.karstii,C.plurivorum,C.siamense and C.wuxiense),and two novel species,described here as C.jinshuiense and C.pyrifoliae.Systematic observation of field symptoms showed that there were five different types of symptoms of pear anthracnose in China broadly.Two kinds of symptoms were observed on fruit,namely 1)bitter rot showing big sunken rot lesions(BrL)and 2)tiny black spots(TS);Three symptom types were observed on leaves,namely 1)big necrotic lesions(BnL);2)small round spots(SS);and 3)TS.Analyses of the prevalence of 12 Colletotrichum species revealed that C.fructicola was the most dominant,occurring on P.pyrifolia and P.bretschneideri in all surveyed provinces except in Shandong,where C.siamense was dominant.In contrast,only C.siamense and C.fioriniae were isolated from P.communis,with the former being dominant.Thirteen representative Colletotrichum strains(one from each species except two from C.fructicola related to two different symptom types)were selected to fulfill Koch's postulates with a spore suspension on detached leaves of P.pyriforia cv.Cuiguan and fruits of P.bretschneideri cv.Huangguan.From these data it was revealed that many strains caused obvious lesions on fruits(or leaves)under wounded conditions but not under unwounded conditions.The results showed that most of the Colletotrichum spp.were weak parasites and had typical latent infection characteristics,and the latent period of 12 Colletotrichum spp.were different.Moreover,C.fructicola caused TS symptoms on leaves and fruits under unwounded conditions,while it caused rot symptoms on fruits or necrosis lesions on leaves under wounded conditions;the BnL symptoms on leaves could also be induced by C.fructicola isolates,if these isolates were isolated from leaves showing BnL symptoms,indicating C.fructicola have two pathogenic types.Furthermore,a total of 43 representative strains were chosen for virulence assessment on detached fruits of P.bretschneideri cv.Huangguan using mycelium plugs under wounded conditions.The results showed that the Colletotrichum species/isolates showed broad diversities in their pathogenicity and aggressiveness.It indicated that there was significant pathogenic differentiation between Colletotrichum species/isolates from different regions and pear species in China.2.Simultaneous transcriptome analysis of the pathogen C.fructicola and its host pear leaves after inoculation.RNA-Seq of the pear leaves was performed at different infection stages(quiescent and necrotrophic stages)by the two strains(PAFQ31 and PAFQ32)of C.fructicola showing different phynotypes.Totally,7.01–11.29 Gb of clean bases with high quality were obtained from each treatment,and the base mass values Q20 and Q30 met the requirements of high quality.Both transcriptome data of C.fructicola and its infected pear leaves were respectively analyzed using DESeq2.Totally,5461 and 10611 genes were differentially expressed(twofold)between the strain PAFQ31 and PAFQ32 compared with pathogen control,respectively;and 4282 and 5834 genes were differentially expressed(twofold)in the pear leaves infected by strain PAFQ31 and PAFQ32,respectively,compared with control leaves.According to the heat map analysis of differentially expressed genes(DEGs),although DEGs in different infection stages of pathogen C.fructicola overlapped,but they mainly showed distinct stage-specific expression.The expression pattern of DEGs in the host P.pyrifolia in the quiescent stage was similar to that in the control group,but significantly different from that in the necrotrophic stage,indicated that the host P.pyrifolia had not activate multiple defense measures against C.fructicola until the pathogen started necrotrophic stage.In addition,there were significant differences of DEGs in P.pyrifolia during the necrotrophic stage of strains PAFQ31 and PAFQ32,indicating that the defense strategies adopted by the host in response to pathogen colonization might be different.Both GO classification and KEGG enrichment analysis of the DEGs showed that C.fructicola had more active synthesis,degradation and energy metabolism in the pathogen necrotrophic stage than quiescent stage.Pear leaves did make defence to C.fructicola by activating a multi-stage immune system.3.Determination of the host defense strategies and prediction of C.fructicola candidate effectors.Through analyses of different metabolic pathways in the pear leaves infected by C.fructicola,it was clear that PTI,ETI,plant hormone immunity,flavonoid and phenypropanoid metabolism were all involved into the resistance to C.fructicola in pear leaves.In addition,various types of transcription factors were involved in the regulation of immune responses during C.fructicola infection.By comparing the defense strategies of host against the infection of two different pathogenic strains of C.fructicola,we found that there were clear differences in the above defense pathways,and the difference in hormone signaling pathway was the most evident.Of which,SA signaling pathway plays an important role for the resistance to PAFQ32 in pear leaves,but JA and ET signals were largely inhibited.However,pear leaves mainly rely on the cross talk of ET,ABA and JA signaling pathways to resist the infection of strain PAFQ31.Candidate effectors were screened by using DEGs of strain PAFQ31 according to the characteristics of the effector.A total of 65 candidate effectors were identified,and 18 of them were significantly upregulated in the process of infection by the strain PAFQ31 on pear leaves.Two significantly up-regulated candidate effectors(CfCP1 and CfE1)were selected for sequence analysis,and they were conservative among different Colletotrichum species and widely exists in ascomycetes.Quantitative real-time PCR proved that both CfCP1 and CfE1 genes were significantly up-regulated during the infection period of C.fructicola,indicating that they may participate in the process of the C.fructicola infection on pear leaves.