Screening of Soil Antagonistic Bacteria for Watermelon Fusarium Wilt

[Objective] The aim of this study was to screen bacteria strains with stable antagonistic effect against watermelon fusarium wilt from soil and investigate the biological control of watermelon fusarium wilt by applying the antagonistic bacteria strains into soil.[Method] Actinomycete strains,fluorescent bacteria strains and bacillus strains were isolated from soil samples by the dilution-plate method,then its resistance was screened respectively by the improved confront culture method after colonies were purified.Finally,bacteria strains with better antagonistic effect were identified.[Result] 29 bacteria strains with stable antagonistic effect against watermelon fusarium wilt were screened from 39 soil samples,which contained 15 fluorescent bacteria strains,5 bacillus strains and 9 actinomycete strains.Furthermore,three antagonistic bacteria strains of FM2,FM3 and FM4 with the strongest antagonism were identified primarily.[Conclusion] According to cultural characteristics,morphological observation,biochemical and physiological tests,FM2 belongs to bacillus subtilis,while FM3 and FM4 belong to micrococcus.

Analysis of Rhizosphere Microbial Community Structure in Different Pathogenesis Stages of Watermelon Fusarium Wilt

High-throughput sequencing technique was applied to analyze the microbial community structure of rhizosphere soil at different stages of watermelon fusarium wilt to find out the difference of dominant microbial community in rhizosphere during the occurrence of watermelon fusarium wilt.Illumina-Hiseq high-throughput sequencing platform was used to sequence 16S and ITS rDNA in rhizosphere soil.The soil was named CK1 before planting,CK2 at peak stage and CK3 at stable stage.The results showed that the soil bacterial diversity was in the order of CK1>CK3>CK2,indicating no significant difference between CK1 and CK3 and a significant difference between CK2 and CK1,CK3.At the genus level,the dominant bacteria were Mizugakiibacter(21.9299%),Rhodanobacter(5.0933%),and Lactobacillus(3.1921%).The diversity of soil fungi was in the order of CK1>CK3>CK2,all showing significant differences.At the genus level,the dominant fungus was Lysurus(54.4601%),Papulaspora(12.4252%),Acrophialophora(3.1729%).The results showed that the diversity and abundance of bacteria and fungi in rhizosphere soil decreased during the peak period of watermelon fusarium wilt.With the gradual stabilization of the disease,the diversity and abundance of bacteria and fungi in rhizosphere soil recovered to a certain extent.

Control Effects of Biological Fertilizer on Watermelon Fusarium wilt

With conventional fertilization as the control, the control effects of biological fertilizer and general organic fertilizer on watermelon Fusarium wilt were studied in the paper. The results showed that applying biological fertilizer could effectively reduce the incidence rate of watermelon Fusarium wilt and significantly improve the quality of watermelon, while application of general organic fertilizer had no obvious control effect on watermelon Fusarium wilt.

Purification of Antifungal Protein from Biocontrol Strain XG-1 against Fusarium Wilt of Watermelon

[Objective] This study aimed to isolate and purify the antifungal protein against Fusarium oxysporum f. sp niveum （FON） from Bacillus subtilis XG-I. [Method] The crude protein was obtained by using fractional precipitation with am- monium sulfate, then chromatography using both DEAE-Sepharose FF anion ex- change and Sephacryl S-100 gel filtration columns was adopted for further purifica- tion of the protein, followed by MALDI-TOF-MS analysis of the structure of the at- tained protein which was antagonistic to FON. [Result] The result from MALDI-TOF- MS analysis suggested that the antifungal protein extracted from strain XG-1 was highly homologous to the flagellin （gi114278900） deriving from B. subtilis, with a pro- tein score of 248 and a coverage rate of 63%. It was thus speculated that this an- tagonistic protein was a kind of flagellin with a molecular mass of 30.6 kD. [Conclusion] The results from this study provide theoretical basis for clarifying the mechanisms of the action of strain XG-1 as well as important references for the preven- tion and control of watermelon Fusarium wilt.

基于BSA-seq技术对西瓜抗枯萎病生理小种1的基因定位

西瓜枯萎病是由尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum)引起的土传真菌病害,是西瓜生产的限制因子。本研究以西瓜抗病种质“V13-9-3”和感病种质“W16-1”为亲本,构建了“W16-1”דV13-9-3”的F1、F2群体。苗期人工接种结果显示:F1植株表现抗病,F2群体的抗病∶感病分离比为3∶1,表明西瓜枯萎病生理小种1的抗性遗传受显性单基因控制。利用BSA技术,采用SNP指数和InDel指数法,将西瓜抗枯萎病基因Fon-1定位在1号染色体上2.20 Mb和2.32 Mb区域内,将这2种方法的定位结果取交集,最终将目标抗病基因区域缩小在2.20 Mb之内,该区域包含8个非同义突变基因和1个移码突变基因。利用荧光定量PCR检测这9个突变基因在接种枯萎病菌5 d和10 d后的表达量变化,发现抗病亲本“V13-9-3”中有7个基因的表达量显著高于感病亲本“W16-1”。研究通过结合SNP指数和InDel指数分析可以快速进行基因初定位,为西瓜抗枯萎病的分子机理研究提供理论依据。

西瓜枯萎病对根际细菌多样性的影响

通过系统分析北京地区西瓜根际微生物组,显示了细菌群落结构分布的显著区域性,磷元素和钾元素是影响西瓜根际菌群组成最主要的因子;扩增子测序分析表明,健康植株比发病植株根际土壤群落结构更为稳定,节点微生物更多;网络分析发现了来自5个菌门的19个菌属与镰刀菌属直接相关,其中芽孢菌属与假单胞菌属与镰刀菌属丰度呈正相关。通过对比西瓜健康植株与镰刀菌侵染植株根际细菌组成差异,并构建相关性网络发现了潜在的枯萎病防治菌群,为北京西瓜枯萎病预警和防控奠定了基础。

尖孢镰刀菌西瓜专化型RPA检测方法的建立与评价

[目的]构建了西瓜枯萎病菌——尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)的重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)的快速检测体系,以期为田间西瓜枯萎病的快速检测提供技术支持。[方法]从NCBI网站下载包括西瓜枯萎病菌在内的11种病原真菌的基因组序列,利用SeqHunter 2软件进行生物信息学分析,经比对得到4个FON的特异性基因片段,并对其进行引物设计和筛选。利用聚合酶链式反应(PCR)验证引物的特异性和灵敏度,然后利用建立的RPA检测体系对引物的特异性和灵敏度进行验证,最后进行人工接种FON的西瓜植株的RPA检测与评价。[结果]建立了FON的RPA检测体系,并对DNA恒温快速扩增试剂盒的扩增体系进行最佳反应时间(40 min)和最适反应温度(30℃)的摸索,得到其最佳反应体系的检测下限为100 pg·μL^(-1)。利用DNA恒温快速扩增试剂盒(胶体金试纸条型)进行扩增时,只需在38℃反应12 min,取50μL稀释20倍的扩增产物用胶体金试纸条进行检测,5 min后即可进行结果的判断,检测下限为100 pg·μL^(-1)。[结论]成功构建FON的RPA检测体系,且建立的RPA检测体系能够从温室接种FON之后未显症的西瓜植物组织基因组DNA中检测到FON,可为田间西瓜枯萎病早期的快速检测提供技术支持。

一种设施西瓜高效绿色防控方法的建立

为了给设施西瓜种植中的高效病虫害防控方法的推广和应用提供理论依据,分别从产量、品质及对蚜虫、枯萎病的防效对传统西瓜病虫害防控方法(T1)、高效绿色防控方法(T2)和传统高温闷棚法(T3)种植条件下的设施西瓜进行比较。利用高效绿色防控方法种植的西瓜较其他处理单果质量大、果皮薄、产量高;中心可溶性固形物、边部可溶性固形物含量高,口感清甜;维生素C、可溶性蛋白含量显著提高,营养丰富,品质提高;对蚜虫的持续防控效果好,有效减少了蚜虫的始发生量,较T1减少了15.54%,并可以安全、持续、有效控制蚜虫危害;T2、T3处理控制枯萎病等土传病害的防效可达100%。综上,集成的设施西瓜高效绿色防控方法,较传统防治方法的产量、品质及蚜虫、枯萎病的防效都有显著提升,可大面积推广应用。

万寿菊根的提取物对西瓜枯萎病反应的抗性研究

本文研究了万寿菊根的提取物对西瓜枯萎病菌和西瓜幼苗的影响。结果表明 ,万寿菊根的提取物对供试的西瓜枯萎病菌菌丝生长有明显的抑制作用 ;同时 ,经对西瓜幼苗根和叶的酶活性测定和过氧化物同工酶电泳分析的结果表明 ,该提取物能诱导西瓜保护酶如超氧化物歧化酶 (SOD)和过氧化物酶 (POD)活性提高 。

根际施用微生物有机肥防治连作西瓜枯萎病研究

为了探讨西瓜专用微生物有机肥料（BOF）对西瓜植株枯萎病的防治效果，采用盆栽试验研究了西瓜专用BOF对连作土壤上西瓜植株生长和枯萎病的防治效果以及西瓜枯萎病致病菌的数量分布的影响。结果表明：1）营养钵育苗和移栽土壤中都施用BOF的处理，未发现西瓜枯萎病植株，而对照植株则完全发病；2）施用BOF处理的致病菌（尖孢镰刀菌西瓜专化型）数量在根际土壤中为0.7×103 cfu/g，在土体土壤中为2.7×103 cfu/g，都控制在104 cfu/g数量级以下，而对照处理的数量分别达到了1.17×105 cfu/g和1.1×105 cfu/g；3）与对照比较，营养钵育苗时施用1%的BOF，西瓜苗期（播种后17 d）生物量都显著增加，根系生物量比地上部生物量提高了16.7个百分点（以鲜重计）和24.8个百分点（以干重计）；营养钵育苗和移栽土壤中均施用BOF的处理，西瓜植株（生长67 d）生物量（干重）是对照的1.83倍；在营养钵育苗或移栽时施用BOF的处理，西瓜植株干重两者差异不大，分别是对照的1.28倍和1.27倍。采用营养钵育苗和移栽时都施用BOF的方法种植西瓜，能有效地促进西瓜植株生长，防止西瓜枯萎病发生，克服西瓜连作障碍。

生防菌NH-BS-2000对西瓜枯萎病原菌抑制作用的研究

测定了生防菌NH BS 2000对西瓜枯萎病原菌的抑制作用.结果表明,培养温度对生防菌NH BS 2000的生长有显著影响,35℃培养的生防菌浓度比15℃培养的多54倍,它们对镰刀菌的抑制率分别为74.0%和63.2%,差异显著;浓度对生防菌抑制病原菌作用的影响不显著,生防菌液原液与1000倍稀释液对病原菌抑制率分别为62.0%和64.2%;用生防菌NH BS 2000处理病原菌8d后,抑制率均在62%以上,最高抑制率达80.6%,而且抑制作用的持续性强.说明NH BS 2000菌株具备作为枯萎病生防菌的良好优势.

西瓜枯萎病菌生理小种1抗性基因连锁标记开发

【目的】开发与栽培西瓜枯萎病菌生理小种1抗性基因Fon-1紧密连锁的分子标记,为开展西瓜抗枯萎病分子辅助育种提供技术支撑。【方法】以栽培西瓜抗枯萎病品种Calhoun Gray为父本,感病品种Black Diamond为母本杂交获得的F2分离群体为供试材料,采用BSA法对Fon-1基因进行区间定位。依据11份栽培西瓜重测序信息,获取位于定位区间内的候选SNP位点。针对候选SNP位点设计CAPS/dCAPS标记,通过F2代分离群体和种质资源验证上述标记与Fon-1基因的连锁关系。【结果】通过BSA法,将Fon-1基因定位于1号染色体15 cM区间内。利用候选SNP位点信息,开发3个CAPS/dCAPS标记7716_fon、7419_fon和4451_fon,F2代分离群体以及164份西瓜育种材料验证显示,上述3个标记与Fon-1基因的连锁距离分别为0.8、1.0和2.8 cM。【结论】利用栽培品种Calhoun Gray的抗性遗传机制,开发的3个CAPS/dCAPS标记可以有效区分栽培西瓜对枯萎病菌生理小种1的抗病、感病性,为栽培西瓜品种枯萎病菌生理小种1抗性基因快速应用于栽培品种枯萎病抗性改良建立有效的技术手段。

枯萎病菌对西瓜不同抗感品种丙二醛含量及某些保护酶活性的影响

本文以西瓜为材料 ,研究了西瓜苗期感染枯萎病菌 ( Fusarium oxysporum Schlecht.)后根部细胞内丙二醛含量、超氧化物歧化酶和过氧化氢酶活性的动态变化。结果表明 ,受枯萎病菌侵染的幼苗细胞内 MDA含量的增加比率 ,抗病品种克伦生低于感病品种早花 ;SOD酶活性的下降比率 ,抗病品种克伦生低于感病品种早花 ;CAT酶活性增减的幅度 ,抗病品种克伦生低于感病品种早花。抗病品种克伦生在染病后的短时间内 ( 4 8h左右 )能使细胞内 MDA、SOD酶和 CAT酶的代谢基本恢复到正常状态 ,其自我调节和恢复正常状态的能力显著大于感病品种早花。

Screening for the Strain Highly Producing Antagonistic Substance from Bacillus subtilis B47 by UV Mutagenesis

[Objective] The aim of this study was to breed new strains which have higher inhibitory effects on the pathogens of watermelon fusarium wilt.[Method] The endophytic Bacillus subtilis B47 strain was obtained from tomato stems by UV mutagenesis for two consecutive times,then genetic stability as well as physiological and biochemical properties of mutant strains were studied.[Result] The antibacterial activity of all the three mutant strains F303,F304 and F305 was higher than that of B74 strain.After subculture of 10 successive generations,the antibacterial activity of all the three mutant strains for the pathogens of watermelon fusarium wilt decreased,but the antibacterial activity of F305 strain decreased the least,indicating its best genetic stability among the tested strains.The antibacterial circle diameter of F305 strain was 5 mm larger than that of wild strain B47 under the same condition.The mutant strain F305 was in logarithmic growth phase within 36 h and in stationary phase within 36-96 h,while its optimum growth temperature was 35 ℃.F305 strain could grow in sodium salt with the concentration of 1%-10%,but it grew best at the concentration of 1%.Physiological and biochemical responses of F305 strain were in accordance with those of wild strain B47.[Conclusion] This study lays the foundation for the factorial production of antagonistic substance by B47 strain and new methods of preventing from the pathogens watermelon fusarium wilt.

西瓜内生枯草芽孢杆菌BS211的拮抗活性及盆栽防效

对从西瓜植株体内分离得到的内生拮抗枯草芽孢杆菌BS211的抗菌谱进行了测定;用5种培养基对BS211菌进行培养,测定了这5种BS211菌培养液无菌滤液的抑菌活性;采用硫酸铵沉淀法提取BS211菌抗菌粗提物,并测定了抗菌粗提物的抑菌活性和温度稳定性;运用砂培和盆栽方法进行BS211菌对西瓜枯萎病的防效试验;测定了BS211菌的宿主范围。结果表明:BS211菌对西瓜枯萎病菌等12种病菌都具有强烈的拮抗作用,而且拮抗性能稳定;BS211菌在5种培养基中的生长量没有显著差异而且均能产生抗菌物质,但在不同培养基中产生的抗菌物质的抑菌能力有差异;BS211菌液的硫酸铵沉淀物及上清液对不同病菌的抗性存在差异,说明BS211菌株产生的抗菌物质为复合物;BS211菌抗菌粗提物具有较好的温度稳定性,尤其是硫酸铵沉淀的上清液部分经121℃处理30 m in后活性无明显变化,对番茄青枯病菌仍有强烈的抑菌作用;BS211菌对西瓜枯萎病有显著防效;BS211菌能在辣椒等多种作物体内定殖,有较强的宿主适应性。

两株解淀粉芽胞杆菌对西瓜枯萎病的防治作用及其相关生防因子检测

本试验从西瓜枯萎病生物防治实际需要出发,检测了两株解淀粉芽胞杆菌TR2和CE对西瓜枯萎病的防治作用及其相关的生防因子。平板对峙试验显示生防菌TR2和CE对西瓜枯萎病尖孢镰刀菌Xg-1、Xg-2和腐皮镰刀菌Xg-3的生长有较强的抑制作用,其中TR2对Xg-1的抑菌效果最好,抑菌带宽度为4.7 mm,CE对Xg-3的抑菌效果最好,抑菌带宽度为5.0 mm,抑菌圈边缘菌丝出现变细、扭曲、个别细胞膨大成球形等畸形现象。幼苗接种试验表明生防菌TR2和CE对西瓜枯萎病的发生具有防治效果,其中TR2对Xg-1在第15 d时防病效果最好,病情指数明显降低,其防治效果达到60%;TR2和CE对Xg-3在第3 d时表现很好的防病效果,发病率和病情指数都明显低于对照,防治效果可达63.6%。采用结晶紫染色、钼锑抗比色法、四苯硼钠法和透明圈检测等方法检测TR2和CE的相关生防因子,发现TR2和CE均可以形成生物膜、分解有机磷、无机磷和钾矿石,并且具备产生蛋白酶、淀粉酶和纤维素酶的能力。采用水培法发现TR2和CE的解磷解钾能力可以促进西瓜幼苗根和茎的生长。结果表明生防菌TR2和CE可以控制西瓜枯萎病的发生、形成生物膜在西瓜根系定殖,促进西瓜幼苗生长,是具有开发潜力的生防菌株。

西瓜与旱作水稻间作改善西瓜连作障碍及对土壤微生物区系的影响

【目的】探讨西瓜与旱作水稻间作减缓西瓜连作障碍的可行性,并从微生物多样性与连作障碍关系角度阐释该方法的作用机理。【方法】盆栽方法研究西瓜与旱作水稻间作改善西瓜连作障碍的效果,传统微生物计数及磷脂脂肪酸(PLFA)法研究西瓜根际土壤微生物区系。【结果】西瓜连作土壤上单作西瓜其枯萎病发病率为66.7%,死亡率为44.4%,与旱作水稻间作后西瓜生长正常。西瓜定植30d后,间作处理西瓜根际的尖孢镰刀菌数量显著低于单作;50d后,间作西瓜根际土中的真菌数量显著低于单作,而细菌、放线菌及总微生物数量均显著高于单作。磷脂脂肪酸(PLFA)法分析土壤微生物多样性发现,单作处理的西瓜根际土壤中真菌数量较多,细菌数量较少,而且主要是革兰氏阳性菌,而间作西瓜根际则含有较多的革兰氏阳性菌、革兰氏阴性菌和放线菌。间作处理西瓜体内的丙二醛(MDA)含量及保护性酶类如根系及叶片中的过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)和根系中的苯丙氨酸解氨酶(PAL)活性均显著低于单作西瓜。【结论】西瓜与旱作水稻间作条件下西瓜根际尖孢镰刀菌数量显著降低,根际微生物区系向细菌与放线菌占主导的趋势发展,有效防止了西瓜枯萎病的发生,改善了西瓜连作障碍。

西瓜抗枯萎病相关EST-SSR的信息分析

对西瓜枯萎病菌生理小种1诱导的抑制差减杂交cDNA文库测序获得的4 000条EST进行分析,经过前处理后拼接得到1 487条unigene序列,全长为759 kb。在其中978条unigene序列中共检索出2 136个EST-SSR,出现频率为53.4%。EST-SSR的平均分布距离和平均长度分别是1/0.36 kb、19.59 bp。EST-SSRs中的重复单元以二核苷酸和三核苷酸重复为主,二者在总EST-SSRs中的出现频率为98.08%。GA/TC和GAA/TTC是二、三核苷酸中的优势重复类型,分别占二、三核苷酸重复的25.78%和12.97%。西瓜抗枯萎病相关EST资源的SSR信息分析为进一步建立西瓜EST-SSR标记和探索其在西瓜基因组学研究中的应用奠定了基础。

中生菌素和氨基寡糖素对西瓜枯萎病防治试验

通过室内和田间试验 ,评价了中生菌素和氨基寡糖素与多菌灵混用对西瓜枯萎病的防治效果。结果表明 ,中生菌素室内抑菌效果明显 ,与多菌灵混用的田间防效和西瓜增产率分别为 84.12 %和 10 4.6 1%,均极显著优于其他药剂处理。氨基寡糖素诱导西瓜抗枯萎病效果明显 ,盆栽试验发病轻 ,植株健壮 ,根系发达 ,田间试验防病增产效果显著。

西瓜品种枯萎病抗性的遗传研究

选用7份对西瓜枯萎病抗性不同的材料作亲本,采用Griffing双列杂交方法II对西瓜枯萎病抗性遗传规律进行研究。试验结果表明,西瓜对枯萎病抗性由隐性多基因控制,其遗传符合加性-显性模型,加性效应比显性效应更为重要,且感病对抗病表现为部分显性,显性方向指向增效,隐性基因为减效,在所有亲本中所携带的隐性基因比显性基因多。且不同西瓜抗性材料的杂交组合所表现的遗传规律不一致:Calhoun Gray、Sugerlee对枯萎病1号生理小种的抗性遗传是呈基因互作的单基因显性性状遗传;Charleston Gray和欣抗♀、红5-2对枯萎病1号生理小种的抗性遗传则很复杂,不能用简单的显隐关系来解释。