林业科学
林業科學
임업과학
SCIENTIA SILVAE SINICAE
2015年
4期
116-125
,共10页
杨蕾%梁军%周国英%倪杨%吕全%张星耀
楊蕾%樑軍%週國英%倪楊%呂全%張星耀
양뢰%량군%주국영%예양%려전%장성요
土壤微生物%杨树溃疡病%木贼镰刀菌%拮抗活性%生防菌株
土壤微生物%楊樹潰瘍病%木賊鐮刀菌%拮抗活性%生防菌株
토양미생물%양수궤양병%목적렴도균%길항활성%생방균주
soil antagonistic microorganisms%poplar canker%Fusarium equiseti%antagonistic activity%bio-control strain
【目的】杨树溃疡病是中国杨树人工林重大生物灾害之一，采用生物防治的方法控制杨树溃疡病是持续有效的手段。本研究旨在从杨树林地土壤中分离出对杨树溃疡病菌有良好生防效果的拮抗微生物。【方法】以杨树溃疡病病原菌葡萄座腔菌为靶标，进行土壤中生防微生物的分离、筛选和鉴定。土壤微生物的分离采用稀释平板涂布法；拮抗微生物的筛选分为初筛、发酵液复筛和拮抗菌株离体复筛3步，初筛采用平板对峙法，拮抗菌发酵液复筛选择三明治法和改良琼脂扩散法相结合的方法，最终通过离体组织防治效果测定确定目的生防菌株；生防菌株的鉴定采用形态学观察和分子生物学鉴定相结合的方法。【结果】在河北省廊坊市和河南省原阳县的杨树林地中按上、中、下3层共采集48份土样，从土样中共分离得到微生物259株，其中细菌122株；放线菌106株；真菌31株，各土层微生物数量规律整体符合上层＞中层＞下层；通过平板对峙法初筛选择出8株抑菌带直径＞4 mm的细菌和放线菌以及8株拮抗菌菌落直径＞40 mm的真菌；再通过发酵液三明治法和改良琼脂扩散法复筛，筛选出8株对杨树溃疡病有良好抑制作用的拮抗菌，分别是细菌TYZ1B3和YX5B1，真菌LS10F1，LX5F1，LZ10F1，LS6F1，LX6F2和 TLZ2F2；最终通过离体组织防治效果测定，从这8株拮抗菌株中筛选出1株土壤生防真菌LX6F2，其对杨树离体组织溃疡病的防治效果可达76.04%；经过形态学观察，菌株LX6F2的菌落、菌丝及孢子形态符合镰刀菌的特征，通过 rDNA-ITS序列及其系统发育分析，测得生防菌株 LX6F2的序列长度为563 bp，序列登录号为 FR872729.1，其与编号为 JN038467的木贼镰刀菌相似度高达100%，从而鉴定该菌株为木贼镰刀菌。【结论】该菌株的发现为杨树溃疡病的生物防治提供了新的原材料，对杨树溃疡病的可持续控制具有重要意义，可在后续研究中将进一步对其抑菌机理、有效拮抗成分及菌剂研制等进行研究。
【目的】楊樹潰瘍病是中國楊樹人工林重大生物災害之一，採用生物防治的方法控製楊樹潰瘍病是持續有效的手段。本研究旨在從楊樹林地土壤中分離齣對楊樹潰瘍病菌有良好生防效果的拮抗微生物。【方法】以楊樹潰瘍病病原菌葡萄座腔菌為靶標，進行土壤中生防微生物的分離、篩選和鑒定。土壤微生物的分離採用稀釋平闆塗佈法；拮抗微生物的篩選分為初篩、髮酵液複篩和拮抗菌株離體複篩3步，初篩採用平闆對峙法，拮抗菌髮酵液複篩選擇三明治法和改良瓊脂擴散法相結閤的方法，最終通過離體組織防治效果測定確定目的生防菌株；生防菌株的鑒定採用形態學觀察和分子生物學鑒定相結閤的方法。【結果】在河北省廊坊市和河南省原暘縣的楊樹林地中按上、中、下3層共採集48份土樣，從土樣中共分離得到微生物259株，其中細菌122株；放線菌106株；真菌31株，各土層微生物數量規律整體符閤上層＞中層＞下層；通過平闆對峙法初篩選擇齣8株抑菌帶直徑＞4 mm的細菌和放線菌以及8株拮抗菌菌落直徑＞40 mm的真菌；再通過髮酵液三明治法和改良瓊脂擴散法複篩，篩選齣8株對楊樹潰瘍病有良好抑製作用的拮抗菌，分彆是細菌TYZ1B3和YX5B1，真菌LS10F1，LX5F1，LZ10F1，LS6F1，LX6F2和 TLZ2F2；最終通過離體組織防治效果測定，從這8株拮抗菌株中篩選齣1株土壤生防真菌LX6F2，其對楊樹離體組織潰瘍病的防治效果可達76.04%；經過形態學觀察，菌株LX6F2的菌落、菌絲及孢子形態符閤鐮刀菌的特徵，通過 rDNA-ITS序列及其繫統髮育分析，測得生防菌株 LX6F2的序列長度為563 bp，序列登錄號為 FR872729.1，其與編號為 JN038467的木賊鐮刀菌相似度高達100%，從而鑒定該菌株為木賊鐮刀菌。【結論】該菌株的髮現為楊樹潰瘍病的生物防治提供瞭新的原材料，對楊樹潰瘍病的可持續控製具有重要意義，可在後續研究中將進一步對其抑菌機理、有效拮抗成分及菌劑研製等進行研究。
【목적】양수궤양병시중국양수인공림중대생물재해지일，채용생물방치적방법공제양수궤양병시지속유효적수단。본연구지재종양수임지토양중분리출대양수궤양병균유량호생방효과적길항미생물。【방법】이양수궤양병병원균포도좌강균위파표，진행토양중생방미생물적분리、사선화감정。토양미생물적분리채용희석평판도포법；길항미생물적사선분위초사、발효액복사화길항균주리체복사3보，초사채용평판대치법，길항균발효액복사선택삼명치법화개량경지확산법상결합적방법，최종통과리체조직방치효과측정학정목적생방균주；생방균주적감정채용형태학관찰화분자생물학감정상결합적방법。【결과】재하북성랑방시화하남성원양현적양수임지중안상、중、하3층공채집48빈토양，종토양중공분리득도미생물259주，기중세균122주；방선균106주；진균31주，각토층미생물수량규률정체부합상층＞중층＞하층；통과평판대치법초사선택출8주억균대직경＞4 mm적세균화방선균이급8주길항균균락직경＞40 mm적진균；재통과발효액삼명치법화개량경지확산법복사，사선출8주대양수궤양병유량호억제작용적길항균，분별시세균TYZ1B3화YX5B1，진균LS10F1，LX5F1，LZ10F1，LS6F1，LX6F2화 TLZ2F2；최종통과리체조직방치효과측정，종저8주길항균주중사선출1주토양생방진균LX6F2，기대양수리체조직궤양병적방치효과가체76.04%；경과형태학관찰，균주LX6F2적균락、균사급포자형태부합렴도균적특정，통과 rDNA-ITS서렬급기계통발육분석，측득생방균주 LX6F2적서렬장도위563 bp，서렬등록호위 FR872729.1，기여편호위 JN038467적목적렴도균상사도고체100%，종이감정해균주위목적렴도균。【결론】해균주적발현위양수궤양병적생물방치제공료신적원재료，대양수궤양병적가지속공제구유중요의의，가재후속연구중장진일보대기억균궤리、유효길항성분급균제연제등진행연구。
Objective]Poplar canker is one of the major biological disasters of poplar plantations in China. The biological control method for regulating poplar canker is sustained and effective. The aim of this study is to find and isolate antagonistic microorganisms which have good bio-control effect on poplar canker from the poplar forest soil.[Method]In this study,poplar canker pathogen Botryosphaeria dothidia was used as the target strain,and the bio-control microorganisms in the soil were isolated,screened and identified. The dilution plate coating method was used to separate soil microorganisms. Three procedures were applied to screen the antagonistic microbes,including primary screening,fermentation liquor secondary screening and the inhibitory action in vitro. The dual culture method was used in primary screening,the“sandwich method”and the“improved agar diffusion method”were combined in fermentation liquor secondary screening,and the objective bio-control strain was used ultimately to determine the control effect on poplar canker in vitro tissue. The bio-control strain was identified by combining morphological observation and the the molecular biology method. [Result]Totally 48 soil samples were collected in upper,middle and deeper soil layers of poplar forest in Langfang Hebei and Yuanyang Henan. A total of 259 strains were isolated in the 48 soil samples,including 122 bacteria strains,106 actinomycetes strains and 31 fungi strains. The microbial distribution pattern overall followed as: top layer soil > middle layer soil > lower layer soil. There were 8 bacteria and actinomycetes strains, screened in primary screening,which had antibiological average diameters >4 mm,and there were 8 fungi strains,selected by dual culture,which had colony diameter >40 mm. Eight antagonistic microorganisms strains were selected by fermentation medium secondary screening. They were bacteria strains of TYZ1B3 and YX5B1,and fungi strains of LS10F1,LX5F1,LZ10F1, LS6F1,LX6F2 and TLZ2F2,respectively. Through determination of the control effect in vitro tissue,a bio-control fungus strain,LX6F2,was obtained,and the control effect of the strain could be up to 76. 04%. The LX6F2 feature,including the colonies,hyphae and spores,was matched with Fusarium sp. by morphological observation. Through rDNA ITS sequence and phylogenetic analysis,the LX6F2 sequence length was 563 bp,and the sequence registration number was FR872729. 1. There was 100% similarity between LX6F2 rDNA ITS sequence and the Fusarium sp strain’s sequence which number is JN038467 in GenBank. Based on these results,the strain LX6F2 was identified as Fusarium equiseti.[Conclusion]The discovery of LX6F2 provides a new raw material for biological control of poplar canker. It’s important for sustainable control of poplar canker. In a follow-up study its antibacterial mechanism,effective antagonist compositions,bacterium agent and so on will be investigated.
