Studies On The Pathogenicity And Genetic Diversity In Konnyaku's Soft Rot Bacteria

In this study,fifty-nine strains isolated from soft-rotted konnyaku in serious disease Konjac Field areas in Hubei Province were measured and determinated by the characteristics of pathogenicity and identification of physiological and biochemical experiments And on this basis,using the relevant techniques from the molecular level carried out to identify and groups of soft-rot bacteria analyzed the genetic diversityThe main findings are as follows:1.Pathogenicity of soft-rot bacteria differentiation:59 strains Isolated from amorphopallus soft rot by the activation of culture were inoculated on the healthy Amorphophallus konjac corms of Enshi,the prepared concentration of about 10⁸ cfu/mL suspension of bacteria We slivers the healthy amorphopallus riveri corm diameter 1.5cm and puts in the ball block the antiseptic culture dish,each dish 3,each processing supposes three repetitions With guns drawn pipette 100ul bacteria suspension were inoculated manually puncture,and sterile water as CK,5 days at 28℃moisture training after inoculation to observe and record the results The findings showed that the pathogenicity of 59 strains there is an obvious division,According to their degree of decay after inoculation is divided into high,strong,medium and weak four types,which high pathogenicity has 11,accounting for 18.6%;strong pathogenic of 13,accounting for 23.7%;pathogenicity medium of 11,accounting for 18.6%;The pathogenicity weak has 24,accounting for about 39.1%.Further the different geography origin strain pathogenicity comparison were analyzed,the results show that different strains of Konjac carotovora pathogenicity there is a clear differentiation phenomenon,collected in different regions the pathogenicity different different strains differ in the same area collected.The findings indicated that caused Hubei Province carotovora Konjac virulence of the pathogens has the phenomenon of apparent differentiation with geographical location and there is no direct correlation.2.Konjac soft rot bacteria related physiological and biochemical characteristics: For 59 strains of Gram stain reaction and dissolved potassium hydroxide test results showed that the isolates were gram-negative bacteria Further conventional physiological and biochemical identification of the experimental method were carried out:the salt endurance test,the glucose oxidation and the fermentation test,the sodium malonate test, the citric acid use,the H₂S test,the indole test,the gelatin liquefication test.the starch hydrolisis test.the catalase determination,the 37℃under growth test,the casein hydrolisis and the tyrosine hydrolisis The results showed that the corresponding test strains of the physiological and biochemical indicators of response is different.Reference to "bacteria Berger Identification Manual," the eighth edition of the main physiological and biochemical properties,the initial test to determine for the 59 strains belonging to two different subspecies,one carrot soft rot Erwinia soft rot carrot subspecies(ECC) There are 52,accounting for 88.1%;followed by the sub-species of Erwinia chrysanthemum (ECH) 7 strain,accounted for 11.9%.This result is consistent with the reported soft rot pathogen Konjac unanimously,hus it can be seen,the Hubei Province amorphopallus riveri soft rot's disease germ is mainly caused by Erwinia soft rot carrot subspecies;Next is the Erwinia chrysanthemum subspecies,further explained that the strains causing the amorphopallus soft rot's disease in Hubei Province existence different subspecies3..Konjac soft rot bacteria ITS molecular identification:To amplify 16S-23S rDNA intergenic transcribed spacer(ITS) by PCR and electrophore se through agarose gel,the 59 strains are clustered into two heterogeneous populations of Erwinia carotovora subsp.carotovora,Ecc.and Erwinia chrysanthemi,Ech.This not only the 59 strains of soft rot were identified at the molecular level,also be in accordance with their physiological and biochemical characteristics4.Konjac soft rot bacteria analysis of genetic diversity:The characteristic band patterns of Ecc.and Ech.could be clearly distinguished by repetitive element-polymerase chain reaction(rep-PCR,BOX and J 3 primers).And the fingerprinting of Ecc.stains were also different from each other,the ITS-PCR identified between the two groups there are obvious genetic differences.The finding further indicated the soft rot bacteria population causes amorphopallus disease in Hubei Province have the rich heredity multiplicity.From the cluster map of Rep-PCR fingerprints can be seen in the chain a distance of 0.5,the test for 59 strains of Konjac soft rot is divided into two major groups.May see from the cluster chart,each class group all includes the different pathogenicity strains.in the groupâ… Contains two strains,but the two strains pathogenicity is actually different, the SS4 strain belongs to the pathogenicity strong strain,but WH2 belongs to the pathogenicity weak strain.Class groupâ…¡also displays the similar situation,contains pathogenicity strong strain EBN3,EXH2,EJG1,also has pathogenicity medium strain EJJ1,EJH3,EXS2,meanwhile has pathogenicity weak strain.EBN1,EBN2,EBN4, EXHI,EXY1 But in the chain-like distance is 0.48 time,the test for 59 strains of Konjac soft rot is divided into 4 group groups,each group of group strain pathogenicity also has the very obvious differentiation.But in the big cluster group,the pathogenicity closely strain gathers usually in the sub-cluster group.