Isolation And Identification Of Three Pathogenic Fungi From Pineapple Leaves In Hainan Province And Preliminary Study On Antibacterial Effects Of Two Biocontrol Bacteria

Pineapple,also known as bromeliad,is a perennial monocotyledon herbaceous fruit tree of the genus Ananas from family Bromeliaceae,which has high edible and medicinal value.And in recent years,pineapple leaf disease has occurred seriously in Hainan province.In order to clarify the pathogen and provide a basis for prevention and treatment,relied on the diseased pineapple leaf samples from various regions of Hainan province,this study first obtained the suspected pathogens by using tissue isolation method,and confirmed them by Koch's rule,next identified the pathogens based on their morphological and molecular biological characteristics,then analyzed the genetic diversity of Lasiodiplodia theobromae spp on multi-gene level,finally controlled the pathogens preliminarily with two kinds of crude N-butanol extracts from biocontrol bacteria HAB-2 and HN-2.This study results are as follows:1.After the tissue isolation of diseased pineapple leaf samples from 16 regions of Qiongzhong,Ledong,Chengmai,Danzhou,Changjiang,Sanya,Wanning,Haikou,Dongfang,Baisha,Qionghai,Ding'an,Wuzhishan,Baoting,Tunchang and Lingao in Hannan province,more than 80 isolates of fungi were obtained,and 42 isolates of pathogens were confirmed by Koch's rule.Then according to the source,colony morphology,pathogenicity,etc.,1-2 representative pathogens were selected in each region,finally,18 representative pathogens were obtained.They were identified by morphology and molecular characteristics as 2 genera,3 species.That is 16 isolates of Lasiodiplodia theobromae,1 isolate of Lasiodiplodia pseudotheobromae,and 1 isolate of Curvularia eragrostidis.Among them,the L.pseudotheobromae is a new pathogen of pineapple leaf spot disease.2.The inhibitory effect on HNLD02(L.theobromae),HNTC001(L.pseudotheobromae)and HNLG02(C.eragrostidis)of crude N-butanol extracts from HAB-2 and HN-2 were tested at two concentrations(10 mg/mL,50 mg/mL).The result showed that the crude extract of HAB-2 had lower inhibitory effect on two species of Lysiodiplodia spp.however the inhibitory effect on C.eragrostidis was significant,and the width of inhibiton zone reached 3.96±0.71 mm when 10 mg/mL,5.91±0.20 mm when 50 mg/mL.Meanwhile,the inhibitory effects of the crude extract of HN-2 on the three pathogens were quite significant.The widths of inhibiton zone at L.theobromae,L.pseudotheobromae and C.eragrostidis reached 2.97±0.48 mm?1.65±0.27 mm ?5.01±1.00 mm,respectively,when 10 mg/mL,5.63±1.06 mm,4.54±0.66 mm and 6.74±0.62 mm,respectively,when 50 mg/mL.After comparison,the inhibitory effects of the crude extracts of the two biocontrol strains on the two species of Lasiodiplodia were significantly different,while the inhibitory effects on C.eragrostidis were not(P=0.21>0.05 when 10 mg/mL,P=0.09>0.05 when 50 mg/mL).Therefore,the HN-2 strain has a good potential for safe and effective control of pineapple leaf spot.3.Based on the phylogenetic tree of ITS,TUB2,EF-1?,GAPDH,CHS-1 and ACT combined sequences,the genetic diversity of the 16 isolates of L.theobromae from 16 different regions of Hainan Province showed that this population divided into three groups: HNWN02,HNBT04,HNQH02,HNBS01,HNHK01,HNCJ02,HNDZ01,HNCM02 and HNWZS03 as one group,mainly concentrated in the middle part of Hainan Island;HNTC03 and HNLG04 were in a group,at the north-central part of Hainan Island;HNQZ003,HNLD004,HNSY02,HNDF01 and HNDA03 were in one group,mainly concentrated in the southwest of Hainan Island.