高毒力昆虫病原线虫共生细菌菌株的筛选及其杀虫活性的检测

昆虫病原线虫共生细菌是寄生在昆虫病原线虫肠道的一种细菌,二者互惠共生。实验采用6个不同种的菌株为筛选材料。共生细菌菌株的培养液经85%饱和度的(NH₄)_2SO4盐析,浓缩冻干得到杀虫粗提物。以粗提物注射大蜡螟Galleria mellonella、饲喂玉米螟Ostrinia furnacalis和棉铃虫Helicoverpa armigera,发现Xenorhabdus nematophilus D43、X.bovienii A54、Photorhabdus luminescens HZL和CB-8等4个菌株发酵液的粗提物对昆虫有高的血腔毒性,菌株A54对昆虫又有高的胃毒效果。由此确立A54为高毒力的菌株,其杀虫活性表现为：注射大蜡螟48 h的死亡率为80%,96 h为93.3%;粗提物饲喂玉米螟,72 h死亡率为53.3%,120 h死亡率为100%;饲喂棉铃虫,72 h死亡率为80.1%,120 h死亡率为90%。杀虫粗提物经DEAE-52柱层析分离,得到一个穿透峰和三个盐的梯度洗脱峰,其中穿透峰对昆虫有很好的胃毒效果,但没有血腔毒性;三个盐峰均有很高的血腔毒性,但没有胃毒作用。穿透峰样品饲喂2龄、3龄棉铃虫也有很好的杀虫活性,96 h 2龄棉铃虫的死亡率为65%,3龄棉铃虫的死亡率为30%;处理96 h的棉铃虫同处理前相比体重下降,未死棉铃虫体重明显低于对照。     

昆虫病原线虫共生菌杀虫毒素研究进展

对昆虫病原线虫共生菌杀虫毒素的种类、与口服毒性有关的杀虫毒素以及口服毒性与杀虫毒素基因的关系等研究进展进行了综述,并对未来的研究方向提出了作者的见解。     

Bio-activity of fungal culture filtrates against root-knot nematode egg hatch and juvenile motility and their effects on growth of mung bean (Vigna radiata L. Wilczek) infected with the root-knot nematode,Meloidogyne incognita

Culture filtrates of selected soil fungi, namely Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Penicillium vermiculatum and Rhizopus nigricans exhibited variable response to egg hatching and mortality of the root-knot nematode, Meloidogyne incognita. Higher concentrations of the culture filtrates of all the fungi inhibited egg hatching and proved to be toxic to the juveniles of M. incognita. In addition, development of the gall and multiplication of M. incognita were also found adversely affected in varying degrees on all the plants of Vigna radiata treated with the filtrates. The culture filtrate of A. niger showed highest toxicity to the nematode than those of any other fungus tested. Soil drench application of the culture filtrates gave better seedling growth and least nematode multiplication in comparison to seed soaking treatment.     

Efficacy of Ricinus communis Fruit Meal with and without Bacillus Species on Suppression of Meloidogyne incognita and Growth of Tomato

The efficacy of ground Ricinus communis fruit, with and without Bacillus species, was tested on suppression of Meloidogyne incognita, growth of tomato, soil electrical conductivity (EC) and soil pH. At harvest, 85 days after applying Ricinus organic amendment, Ricinus and initial population nematode density (P_i), accounted for 64–74% of the total treatment variation (TTV) in final population nematode density (P_f) and 53–89% of the TTV in dry shoot weight. Ricinus, P_i and electrical conductivity accounted for 49–69% of the TTV in fresh fruit weight, whereas Ricinus, P_i and Bacillus accounted for 65–82% of the TTV in plant height. Because Bacillus×Ricinus interaction was not significant (P > 0.05) for any variable measured, the efficacy of Ricinus fruit meal was independent of microbial activities.     

M60家族肽酶参与调控蜡质芽胞杆菌AR156防治南方根结线虫病的作用机制

【背景】南方根结线虫(Meloidogyne incognita)寄主范围广泛,能侵染茄科、豆科、葫芦科等蔬菜,对世界农业生产造成了严重危害,每年造成的损失可高达数百亿美元。蜡质芽胞杆菌(Bacillus cereus) AR156是一种革兰氏阳性杆状细菌,对于南方根结线虫引起的土传病害有较好的防治效果,并且已经完成了全基因组测序。【目的】了解AR156对南方根结线虫的生防机理,寻找与生防功能相关的基因,进行遗传功能基因的分析。【方法】构建蜡质芽胞杆菌AR156 miniTn10随机插入突变体文库,通过对南方根结线虫致死率和温室防病试验进行筛选,找到与AR156生防能力相关的突变体,鉴定相关功能基因,并对产蛋白酶活性、定殖能力、生物膜形成和游动性等方面进行检测。【结果】与AR156野生型相比,转座子插入位点在M60家族肽酶的突变体BC41产蛋白酶活性下降,植物根围定殖能力减弱,生物膜形成能力和游动性受到影响,从而导致其防治南方根结线虫的能力降低。【结论】通过对生防相关功能基因的分析,初步明确M60家族肽酶在蜡质芽胞杆菌AR156防治南方根结线虫的过程中发挥重要作用。     

Isolation and screening of fungi associated with eggs and females of root-knot nematodes and their biocontrol potential against Meloidogyne incognita in Bangladesh

A total of 297 fungal isolates belonging to 20 genera and 33 species were isolated and identified from eggs and females of Meloidogyne spp. in Bangladesh. The predominant genera were Fusarium, Aspergillus and Penicillium; and the significant ones were Purpureocillium, Trichoderma and Pochonia. The 24 well tissue culture plate screening technique was applied for pathogenicity tests against Meloidogyne incognita in vitro. The average percentages of egg parasitism, egg hatch inhibition and juvenile mortality varied significantly and were ranged from 8.2 to 64.9% (p = 0.05), 24.8 to 72.4% (p = 0.05), and 2.3 to 33.1% (p = 0.05), respectively. Two isolates of Purpureocillium lilacinum (PLSAU 1 and PLSAU 2) and one isolate of Pochonia chlamydosporia (PCSAU 1) reduced more than 60% average root galls of tomato, eggplant and cucumber in greenhouse experiments. This is the first investigation of fungi associated with nematodes in the country and their biological control potential against M. incognita.     

紫球藻的培养与利用（综述）

紫球藻为单细胞红藻,其细胞富含红藻多糖、多不饱和脂肪酸(主要为AA和EPA）及藻胆蛋白等高价值生物活性物质,在医药和精细化工领域有广泛的应用前景。本文综述紫球藻的生物学特性、生物活性物质、培养与培养特性等方面的研究概况。     

蓖麻提取物和淡紫拟青霉对南方根结线虫的防治作用

通过杀线活性测定及盆栽试验,研究了蓖麻提取物和淡紫拟青霉(Paecilomyces lilacinus)对南方根结线虫(Meloidogyne incognita)的杀线活性及防治效果.结果表明:蓖麻碱不影响淡紫拟青霉孢子的萌发.蓖麻碱和淡紫拟青霉均具有较强杀线活性,蓖麻碱处理对南方根结线虫的卵孵化抑制率和二龄幼虫死亡率分别达61.7%和59.2%,显著高于对照处理;蓖麻碱和孢子液复合处理接种南方根结线虫的番茄苗后,植株平均根结数为15±3,显著低于对照的平均根结数37±2,株高、鲜重和根长增长率分别比对照提高38.5%、44.0%和57.0%.说明蓖麻提取物和淡紫拟青霉能减轻线虫危害,对番茄南方根结线虫病控制效果明显.     

Biological control of Meloidogyne incognita and Fusarium solani in dry common bean in the field

Meloidoyne incognita (root-knot nematode) and Fusarium solani (root-rot pathogen) were the common soil-borne pathogens and cause severe damage to bean plants in newly reclaimed sandy soil in Nubaryia district, Behera Governorate, Egypt. The antagonistic effects of Trichoderma album and Trichoderma viride as well as three commercial products namely Rhizo-N^® (Bacillus subtilis), Bio-Arc^® 6% (Bacillus megaterium) and Bio-Zeid^® 2.5% (T. album) were tested against M. incognita and F. solani under naturally infected field conditions. T. album and T. viride highly reduced the frequency (%) population of pathogenic fungi such as Fusarium spp., F. solani and Rhizoctonia spp., than the commercial products. Results indicated that all the tested bio-control agents reduced, significantly, the nematode criteria as evidenced by the number of juvenile (J₂) in soil and number of galls and egg masses on roots of common bean and Fusarium root-rot incidence (%). Rhizo-N^® highly reduced the number of J₂ in soil, while T. album was the best in reducing the number of galls and egg masses in roots. The bio-control agents also increased the plant growth parameters of common bean plants i.e. plant height, plant weight, branch no./plant, pods no./plant, pod weight/plant, pod weight, seeds no./plant, fresh seeds weight/pod, dry seeds weight/pod and dry weight of 100 seeds.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Characterisation of antagonistic soil microbes against Rhizoctonia spp. and Sclerotium hydrophilum

Rhizoctonia solani, R. oryzae-sativae and Sclerotium hydrophilum are responsible for sheath diseases of rice. Soil treatment measures with antagonist microbes have produced good results against various soil-borne fungi. The objective of this study included to find out the potential antagonist microbes from the paddy field soil. Ten different soil samples were collected from the disease-prone area of paddy field soil. Antagonist microbes were identified by 16S rRNA sequence analysis. Subsequent screening of antagonist microbes yield B. subtilis B37 and P. aeruginosa B258 as the potential antagonist microbes to control sheath diseases of rice in Myanmar. Bacillus subtilis strains induced the competition between antagonist and the tested fungi at the margin of the bacterial growth itself. However, P. aeruginosa induced the competition at a long range formed by the antibiotic of antagonist microbe. Bacillus subtilis B37 and P. aeruginosa B258 could be applicable in the control strategy of causal agents of rice sheath diseases in Myanmar.     

Studies on the disease complex incidence by Meloidogyne incognita and Fusarium solani on Poi,Basella rubra

A survey of Poi crop in Ghaziabad (UP) exhibited a disease complex incidence by Meloidogyne incognita and Fusarium solani causing synergistic effect on the host. Paecilomyces lilacinus was found from the egg masses of M. incognita and Aspergillus niger and Aspergillus terreus from the rhizosphere of root-knot infected Poi crop. Paecilomyces lilacinus parasitised the eggs to a greater extent. The level of parasitism was highest (65%) by P. lilacinus while Aspergillus spp. did not colonise the eggs. Fusarium solani which in the present investigation has been established to be pathogenic to Poi plant.     

Biological control potential of plant growth‐promoting rhizobacteria suppression of Meloidogyne incognita on cotton and Heterodera glycines on soybean: A review

Over the past decade, we have seen an increasing market for biopesticides and an increase in number of microbial control studies directed towards plant‐parasitic nematodes. This literature survey provides an overview of research on biological control of two economically important plant‐parasitic nematodes, Meloidogyne incognita (Kofoid & White) Chitwood (southern root‐knot nematode) and Heterodera glycines Ichinohe (soybean cyst nematode) using spore‐forming plant growth‐promoting rhizobacteria (PGPR). In this review, the current biological control strategies for the management of those cotton and soybean nematodes, the mechanism of using BacillusPGPR for biological control of plant‐parasitic nematode including induced systemic resistance and antagonism and the future of biological control agents on management of plant‐parasitic nematodes are covered.     

Suppressive effects of metabolites from Photorhabdus and Xenorhabdus spp. on phytopathogens of peach and pecan

Abstract

Our objective was to determine the suppressive abilities of bacterial metabolites derived from Xenorhabdus and Photorhabdus spp. on Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, and Fusicladosporium effusum, which are fungal or oomycete pathogens of pecan, and Monilinia fructicola, a fungal pathogen of peach. In the first set of in vitro assays, when metabolites were compared based on initial bacterial cell count, X. bovienii (SN) metabolites generally exhibited the greatest suppression of phytopathogens and Xenorhabdus sp. (355) the least with Photorhabdus luminescens (Hb) and Xenorhabdus nematophila (All) being intermediate. In a second set of in vitro assays, in which metabolites were compared at 50 mg per ml acetone, P. luminescens (VS) exhibited greater suppression than P. luminescens (Hb), Photorhabdus sp. (MX4), X. bovienii (SN), and Xenorhabdus sp. (3 – 8b). In in vivo tests, 6 or 12% dilutions of X. bovienii (SN) or P. luminescens (Hb) metabolites caused 90 – 100% suppression of P. cactorum lesions on pecan leaves with only slight phytotoxicity. No phytotoxic effects were observed in detached peach leaves at dilutions up to 25%. Metabolite treatments, derived from X. bovienii (SN) and P. luminescens (Hb), were also tested for suppression of F. effusum sporulation in detached pecan shoots. Reductions in sporulation caused by bacterial metabolites were similar to those following treatment with two chemical fungicides, dodine and fenbuconazole; a third chemical triphenyltin hydroxide had no effect. Further research is warranted to determine if fungal or oomycete incited diseases in pecan and peach can be controlled with metabolites of Xenorhabdus spp. and Photorhabdus spp.     

银胶菊叶和花提取物对南方根结线虫的毒杀活性比较

为进一步明确银胶菊(Parthenium hysterophorus L.)的杀线虫活性,对银胶菊叶和花的不同溶剂提取物、甲醇提取物的不同萃取物以及甲醇提取物碱水层的不同极性组分对南方根结线虫(Meloidogyne incognita Chitwood)的杀虫活性进行了检测,并对不同提取物、萃取物和萃取组分进行了生物碱的定性分析.结果表明:银胶菊叶和花的蒸馏水、甲醇、乙酸乙酯和石油醚提取物的得率分别为24.5％和20.3％、19.6％和10.9％、6.8％和7.7％、2.0％和2.7％,其中,叶和花的蒸馏水和甲醇提取物的杀线虫活性均较强,而石油醚提取物的杀线虫活性最弱.用质量体积分数1.0％和0.5％的叶和花蒸馏水提取物分别处理24和48 h后试虫的校正死亡率均达到100.00％;用质量体积分数1.0％和0.5％的叶和花甲醇提取物处理48 h,试虫的校正死亡率均大于90％.叶和花甲醇提取物的碱水层、三氯甲烷Ⅰ层和Ⅱ层萃取物均具有一定的杀线虫活性,其中,用质量体积分数1.0％的花和叶碱水层萃取物以及花的三氯甲烷Ⅰ层萃取物分别处理48 h,试虫的校正死亡率均为100.00％,而三氯甲烷Ⅱ层萃取物的杀线虫活性最弱.银胶菊叶和花甲醇提取物碱水层的11个不同极性组分(A1～A11)也表现出不同程度的杀线虫活性,其中,用质量体积分数0.2％和0.1％花的A2[溶剂为V(三氯甲烷)∶V(甲醇)=10∶1]和A7[溶剂为V(三氯甲烷)∶v甲醇)=1∶1]组分以及叶的A2和A6[溶剂为V(三氯甲烷)∶V(甲醇)=2∶1]组分处理48 h后,试虫的校正死亡率均达100.00％,显著高于其他组分.定性实验结果表明:银胶菊叶和花中具有杀线虫活性的提取物、萃取物和萃取组分中均含有生物碱.研究结果说明:银胶菊花的杀线虫活性高于叶片,其毒杀活性不仅与提取部位及溶剂的种类和极性有关,还与提取物浓度及作用时间等因素有关.     

Reproduction of Meloidogyne marylandi and M. incognita on several Poaceae

The susceptibility of 22 plant species to Meloidogyne marylandi and M. incognita was examined in three greenhouse experiments. Inoculum of M. marylandi was eggs from cultures maintained on Zoysia matrella "Cavalier" or Cynodon dactylon x C. trasvaalensis "Tifdwarf". Inoculum of M. incognita was eggs from cultures maintained on Solanum lycopersicum 'Rutgers'. In each host test the inoculum density was 2,000 nematode eggs/pot. None of the three dicot species tested (Gossypium hirsutum, Arachis hypogaea, and S. lycopersicum) were hosts for M. marylandi but, as expected, M. incognita had high levels of reproduction on G. hirsutum and S. lycopersicum. Meloidogyne marylandi reproduced on all of the 19 grass species (Poaceae) tested but reproduction varied greatly (P = 0.05) among these hosts. The following grasses were identified for the first time as hosts for M. marylandi: Buchloe dactyloides (buffalograss), Echinochloa colona (jungle rice), Eragostis curvula (weeping lovegrass), Paspalum dilatatum (dallisgrass), P. notatum (bahiagrass), Sorghastrum, nutans (indiangrass), Tripsacum dactyloides (eastern gamagrass), and Zoysia matrella (zoysiagrass). No reproduction of M. incognita was observed on B. dactyloides, Cyndon dactylon (common bermudagrass), E. curvula, P. vaginatum (seashore paspalum), S. nutans, T. dactyloides, Z. matrella or Z. japonica. Reproduction of M. incognita was less than reproduction of M. marylandi on the other grass species, except for the Zea mays inbred line B73 on which M. incognita had greater reproduction than did M. marylandi (P = 0.05) and Stenotaphrum secundatum (St. Augustinegrass) on which M. incognita and M. marylandi had similar levels of reproduction.     

Effect of resistant and susceptible soybean cultivars on the attraction of egg parasitoids under field conditions

The association of resistant crop varieties and biological pest control has great potential for pest management, as resistant cultivars may affect herbivore population dynamics by interfering with their biology and search behaviour for host plants. In addition, resistant varieties can also affect the efficiency of natural enemies. The aims of this work were to evaluate the influence of the soybean cultivars Dowling (resistant), IAC 100 (resistant) and Silvânia (susceptible) under field conditions on: (i) the abundance of parasitoids in the Platygastridae family and the stink bug Euschistus heros and (ii) the parasitism of stink bug eggs by these natural enemies. There was no difference between cultivars in stink bug distribution in the field. The parasitoids Telenomus podisi, Trissolcus teretis, Tr. urichi and Tr. brochymenae emerged from the sentinel eggs of E. heros, and T. podisi was the most abundant species observed during the reproductive stage R6. The cultivars Dowling and IAC 100 attracted a higher number of Platygastridae parasitoids, T. podisi and Tr. teretis, and the abundance of these parasitoids during the period from R1 to R7 might have being sufficient to control the stink bug population. These results suggest that the cultivars Dowling and IAC 100 have important attributes that should be taken into account in the implementation and development of new cultivars in breeding programmes that aim to obtain plants more resistant to stink bugs and more attractive to natural enemies.     

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae

Aims:  Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes.
Methods and Results:  Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X . szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X . budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite^® XAD 1180, and eluted with methanol : 1  n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX.
Conclusions:  Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions.
Significance and Impact of the Study:  Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P . nicotianae .     

Optimization of different application methods of multi-facial bacterial and fungal antagonists against sheath blight pathogen of rice,Rhizoctonia solani AG1-IA

Relative effectiveness of seven different application methods of five native bacterial and fungal biocontrol agents (BCAs) and one fungicide (azoxystrobin) was evaluated against Rhizoctonia solani, under pot and field conditions on rice cultivar Pusa Sugandha-5. Plants grown in pots infected with R. solani suffered a 30%–49% decrease in plant growth and yield of grain. However, treatment with BCAs reduced the adverse effect of the pathogen but significantly varied with the treatment schemes. Amongst the treatments, soil application (SA) at 20 + 40 days after planting (DAP) followed by foliar application (FA) at 60 DAP was recorded as most efficacious, and reduced the severity of disease by 42–68%, resulting in a 21–36% plant growth promotion and yield enhancement. Treatment comprising SA 20 + FA 40 DAP was next in effectiveness but statistically equal to seed priming (SP) + SA 40 DAP of treatment. Amongst the BCAs, Pseudomonas putida was shown to be the most efficient, trailed by Trichoderma harzianum, P. fluorescens, T. viride and Bacillus subtilis. Field trials under naturally infested fields have also validated the effectiveness of P. putida. The SA 20 + 40 + FA 60 DAP with P. putida and T. harzianum were found quite effective and decreased the disease severity and incidence (40–81%), and improved the grain yield (42–72%). Relatively lower ShB control was recorded with SA 20 + FA 60 DAP, however, it was statistically at par with SA 20 DAP treatment and equal to SA 20 + FA 40 DAP.     

Comparison of Reproduction by Meloidogyne graminicola and M. incognita on Trifolium Species

The reproductive potential of Meloidogyne graminicola was compared with that of M. incognita on Trifolium species in greenhouse studies. Twenty-five Trifolium plant introductions, cultivars, or populations representing 23 species were evaluated for nematode reproduction and root galling 45 days after inoculation with 3,000 eggs of M. graminicola or M. incognita. Root galling and egg production by the two root-knot nematode species was similar on most of the Trifolium species. In a separate study, the effect of initial population densities (Pi) of M. graminicola and M. incognita on the growth of white clover (T. repens) was determined. Reproductive and pathogenic capabilities of M. graminicola and M. incognita on Trifolium spp. were similar. Pi levels of both root-knot nematode species as low as 125 eggs per 10-cm-d pots severely galled white clover plants after 90 days. Meloidogyne graminicola has the potential to be a major pest of Trifolium species in the southeastern United States.