| Amorphophallus Konjac belongs to the perennial herb of Amorphophallus in Araceae,mainly distributed in Yunnan,Guizhou,Sichuan,Chongqing,southern Shaanxi,western Hubei,western Hunan and southwest Henan and other regions.It is the only plant that can synthesizing glucomannan in large quantities,suggesting high economic value.In recent years,konjac industry has developed into a pillar industry leading farmers to get rid of poverty in the central and western regions,at the same time,the continuous expansion of konjac planting area has also intensified the occurrence of diseases.Bacterial soft rot is the most serious disease in Konjac production,found in the whole growth period and storage period of Konjac,causing no harvest in the severely diseased plots,and is characterized by rapid transmission,strong pathogenicity and difficult prevention and control,which severely restricts and threatens the safe and sustainable development of the konjac industry in China.The investigation found that the taxonomic status of soft rot pathogens in Fuyuan and Chuxiong of Yunnan Province and Yibin of Sichuan Province remain be unknown.In this study,the conventional plate line separation method was used to isolate the strains from the soft rot samples of Konjac in the aforementioned regions,and three pathogenic bacteria were obtained by Koch’s rule verification.The identification of the pathogenic bacteria was performed by morphology and molecular biology tests to clarify their taxonomic status,and their biological characteristics,pathogenicity,and host range were analyzed.In addition,the biocontrol effect of Bacillus velezensis on soft rot disease of Konjac was also evaluated.This study will provide theoretical basis for the prevention and control of the disease.The main results of this study are as follows:1.The strains were isolated from soft rot samples konjac in three main konjac planting regions including Fuyuan and Chuxiong of Yunnan and Yibin of Sichuan Province.Three pathogenic strains were obtained by Koch’s rule and named as MY7,MY11 and MY18 respectively.The three strains were translucent round on the LB solid medium,with smooth surfaces and neat edges;they were gram-negative enterobacteria with pectinolytic activity on crystal violet pectate(CVP)medium and peritrichous flagellae,consistent with the morphological characteristics of Pectobacterium.MY7,MY11 and MY18 produced the expected fragment size of about 434 bp using Pectobacterium specific primers Y1/Y2,and exhibited the same band patterns with those of P.aroidearum KC20 on the basis of ITS-RFLP analysis,and were clustered in a P.aroidearum clade by the analysis of 16 S r DNA sequences and pmrA gene sequences,respectively,based on the phylogenetic tree.The genome-wide average nucleotide identity(ANI)values and DNA-DNA hybridization(DDH)values were 98.22% and 83.5% between these three strains and our newly sequenced P.aroidearum KC20,respectively,and between them and reported P.aroidearum PC1 were 98.06% and 81.7% respectively,higher than the threshold(95-96%;70%)of the same species.These results declared that the pathogens MY7,MY11 and MY18 isolated from Fuyuan,Chuxiong and Yibin were identified as P.aroidearum.2.MY7,MY11 and MY18 began to be in logarithmic growth phase after inoculation for 2 h in LB liquid medium,all exhibiting the same growth trend;the optimum growth temperature was 28 °C and the lethal temperature was 42 °C;they could grow at p H 4～10,with the optimum p H of 7;they were sensitive to high acid and alkali environment.In addition,the physiological and biochemical results showed that the three strains could grow normally in 5% and 7% Na Cl and under 37 °C,and could liquefy gelatin,produce no indole and fluorescent pigment on KB’ medium,positive for catalase,oxidase and nitrate reduction,and negative for phosphatase and sucrose reduction,could not use α-methylglucoside,could not produce acid by using D-arabinoside isomaltulose and inulinto.Biolog carbon source utilization results showed that the three strains could use D-cellobiose,gentiobiose,sucrose,β-methyl-D-gluctoside,N-acetyl-D-glucosamine,α-D-glucose,D-mannose,D-fructose,D-Mannitol,glycerol,L-serine,L-aspartic acid,D-saccharic acid,galacturonic acid,citric acid,L-malic acid,methyl pyruvate and bromosuccinic acid;and could not use D-arabitol,L-histidine,L-pyroglutamic acid,quinic acid,propionic acid,dextrin,D-malic acid and acetoacetic acid as the sole carbon source.In addition,MY7 could use formic acid and gluconic acid,different from MY11 and MY18,and MY11 and MY18 both could not use D-trehalose,myo-inositol and acetic acid,different from MY7.MY11 could not use D-galactose and mucic acid.MY18 could not use stachyose,D-raffinose,D-melibiose and L-rhamnose,which were different from other two strains.3.The results of MY7,MY11 and MY18 virulence tests showed that the rotted mass of konjac tubers inoculated with MY7 and MY11 were 0.46 g and 0.55 g,respectively,with the diseased spot length of in vitro stem segment of 1.80 cm and2.30 cm,respectively.However,after inoculation with MY18,the rotting weight of konjac tubers reached 0.66 g,with the diseased spot length of isolated stem segment of 4.80 cm,with the diseased spot length in the stem base of living plants of 0.52 cm,The pathogenicity of MY18 was significantly higher than that of MY7 or MY11.By artificial inoculation,MY7,MY11 and MY18 all could induce obvious soft rot symptoms in 12 plants species,including Carrot(Daucus carota),Avocado(Persea americana),Squash(Cucurbita pepo),Potato(Solanum tuberosum),Garlic(Allium sativum),Chili(Capsicum annuum),Onion(Allium cepa),Green onions(Allium ampeloprasum),Chinese cabbage(Brassica rapa),Celery(Apium graveolens),Lettuce(Lactuca sativa)and Ornithogalum(Ornithogalum dubium).4.The results of the plate antagonism test showed that all tested seven strains of B.velezensis(BPC6,BPC16,W1-1,W2-3,W2-4,W2-7 and E2-4)could inhibit the growth of the pathogen MY18 and produce antibacterial bands.The width of the antibacterial bands of B.velezensis BPC16 and W2-7 were 0.59 cm and 0.54 cm,respectively,showing better antibacterial effect when compared with other five strains.Under the potted condition of greenhouse,the incidence of konjac treated by B.velezensis BPC16 and W2-7 was 56.67% and 63.33% respectively,and the disease index was 28.52% and 34.45% respectively,which were significantly lower than those of CK(water).The control efficacy of B.velezensis BPC16 and W2-7 on konjac soft rot was 43.01% and 31.99% respectively,among which of B.velezensis BPC16 was increased by 9.26% compared with that of zhongshengmycin(33.75%).The results indicated that B.velezensis had potential application in the effective biological control of konjac soft rot. |
