Screening And Identification Of The Casual Pathogen Causing Leaf Yellowing Disease Of Areca Catechu In Hainan

Areca catechu is tropical aiphylliums and one of most important crops,ornamental plants as well as Li ethnic medicine resources in Hainan.The occurrence and rapid spread of Areca yellow leaf disease(YLD)have led to a large reduction of areca production,which seriously affected the healthy development of areca industry.YLD has become the main limiting factor of areca production in Hainan.According to the domestic and foreign reports,the causal agent of YLD is phytoplasma,but the symptoms described by different authors are different.Additionally,no phytoplasma was detected in the YLD samples,so that the pathogen of Areca yellow leaf disease is yet to be identified.In order to find out the main pathogen of YLD in Hainan,a comprehensive investigation was carried out in main plantation area of Areca catechu in Hainan,the pathogenic microorganisms and the causing leaf yellowing symptoms in Areca catechu were exactly described and identified,RNA-seq was applied to reveal the unknown pathogens related with YLD.The main results were obtained as follows:1.Diseases investigation of Areca catechu in HainanThe pathogenic investigation was carried out in the 19 main areca plantations producing areas of in Sanya,Lingshui,Wanning,Qionghai,Wenchang,Ding’an,Tunchang,Qiongzhong and Ledong in Hainan.The results showed that biological factors(pathogenic microorganisms such as leaf diseases and root diseases)and abiotic factors(drought,drug damage,fertilizer damage,improper use of herbicides,etc.)cause leaf yellowing symptoms of Areca catechu in Hainan.Six fungal diseases,including Colletotrichum sp.,Fusarium solani,Curvularia pseudobrachyspora,Pestalotiopsis clavispora,phyllosticha cavendishii,Curvularia lunata and leptoxyphium Fumago,are prevalent in areca palm of Hainan.Bud rot occurs in many areas,leading to bud-rot and even plant death.The viral areca necrotic ring spot disease(ANRSD)and areca necrotic shuttle spot disease(ANSD)are very common,but the damage is not serious.Bacterial leaf spot/stripe spot is infrequent and less harmful.YLD is prevalent and most serious in Hainan areca orchard.The symptoms are evidently different from other diseases;begin with yellowing of leaflet tips of the mid-crown or the lowermost leaves.At the beginning stage,clearly abrupt demarcation of yellow and normal green areas could be observed on the infected leaves,which can be distinguished from physiological yellowing.The chlorosis spreads along the direction of vascular tissue while the midribs remain green,which usually forms a green-yellow border.At the late stage,the yellowing symptom expands to younger leaves and lower leaves,and the green-yellow border on older leaf becomes unclear;newly emerging leaves are stunted;the crown size reduces markedly resulting in “bunchy top” symptom.Additionally,YLD diseased areca palms are more sensitive to drought and cold stresses.2.Identification of new fungal pathogens accosiated with yellowing syndromeTo identify the pathogens causing different yellowing symptoms on areca palm to the previous descriptions,the pathogens were first isolated,and then identified by morphological observation and analyzed by molecular biological methods.The pathogens of gray leaf spot(Curvularia pseudobrachyspora),root rot disease(Fusarium solani)and coal mildew disease(Curvalaria lunata)were first reported on areca plam.Additionally,four races of Colletotrichum sp.(C.gloeosporioides,C.Siamense,C.Fruticola,and C.Camellia)were identified,among which three races were first reported on Areca catechu.This discovery will lay a theoretical foundation for the diagnosis and control of areca diseases in the future.3.Identification of the casual agent of YLD.Although it is reported in the literature that the pathogen of YLD is phytoplasma,But we can not detect phytoplasma in most of the YLD samples.To discover the potential causal agent of YLD,RNA seq and Digital expression spectrum(DEG)method was used to analyze the YLD sample.In this work,The causal agent was searched by digital gene expression analysis(DGE).There are only 819 differentially expressed genes(DEGs)among YLD and health control samples,although 124,053 unigenes were assembled.Nine DEGs showed high expression in both YLD samples but not in health control,of which 6DEGs were matched to Areca palm velarivirus 1(APV1).No unigenes was annotated as phytoplasma,suggesting that phytoplasma did not exist in YLD samples tested.The APV1 may be the casual agent of YLD.To verify the hypothesis,the following assays were carried out.(1)Accossiation of APV1 with YLDTo investigate whether APV1 is associated with YLD,five specific primer sets(YLDV1,YLDV2,YLDV3,YLDV4,and YLDV5)were designed according to the consensus sequences of APV1-HN(KR349464).APV1 was detected by RT-PCR in all76 YLD-affected symptomatic samples collected from eight locations(Table 3),with at least two of five primer sets tested.In addition,eight of the 90 symptomless samples,four each from the YLD epidemic areas of Qionghai and Wanning,tested positive for APV1 by RT-PCR.None of the samples collected from the non-epidemic areas of Haikou and Danzhou was positive for APV1.Thus,the results showed a strong and consistent association between APV1 and YLD.(2)Transmission electron microscopy of APV1 particlesViral particles were purified and analyzed by SDS-PAGE,revealing a 35 k Da bands that is consistent with the predicted size of coat protein of APV1.RNA was isolated from purified viral sample for RT-PCR detection.APV1 was detected in the YLD viral particle sample,suggesting that the viral particle contains APV1.Transmission electron microscopy revealed that the virus particles of APV1 were curved and linear,which is consistent with the morphology of closenoviridae virus.(3)Identification of potential transmission vectors of APV1Many insects were collected from areca palm orchards.PCR results revealed that APV1 was detected in Aleurocanthus spiniferus Quaintance and Pseudococcus sp.,suggesting that these insects ingest APV1 and are the potential transmission vectors of APV1.This work is critical for identification of YLD transmission vector and provides theoretical guidance for the prevention and control of the YLD.In summary,three new fungal pathogens and three new reaces of Colletotrichum related with leaf yellowing syndrome of areca palm were first identified.APV1 was identified as casual agent of YLD in Hainan.APV1 was also detected in Aleurocanthus spiniferus Quaintance and Pseudococcus sp.,suggesting that they are the potential transmission vectors of APV1.Data shown in the work provided a theoretical foundation for diagnosis and control of YLD in Hainan.