A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.     

原生质体融合技术改良植病生防木霉菌株

以产孢量大，对苯菌灵有抗性，对潮霉素B敏感的哈茨木霉菌株T9和产孢量少，对潮霉素B有抗性，对苯菌灵敏感的康宁木霉菌株Tk7a为亲本，进行原生质体融合，筛选获得抗最高浓度杀菌剂的融合子。所有融合子的产孢量均比亲本菌株Tk7a大。融合子按照菌落形态的不同分成4组，大部分融合子的几丁质酶、葡聚糖酶和内切葡聚糖酶的活性比亲本菌株高。融合子对棉花猝倒病菌和小麦全蚀病菌的寄生能力基本等同于亲本菌株；融合子对小麦全蚀病的防效比亲本菌株稍好，其刺激麦苗根部生长的能力与亲本菌株相差不大。两个融合子根际竞争能力比亲本菌株T9强，和亲本菌株Tk7a相当。     

华南地区主要番茄品种对南方根结线虫的抗性评价

通过人工接种方式, 对华南地区番茄主要栽培品种对优势根结线虫——南方根结线虫（Meloidogyne incognita）的抗性进行了评价。结果表明,广东栽培品种年丰、红宝石、新星、福安、粤红玉、穗丰、金丰、益丰、丰顺、大丰顺、满丰和法国品种SAINT PIERRE均高度感病; 而法国品种 PIERSOL VFN高度抗病,根结率为0。根据SAS8.0多重比较分析表明,在F0.01的水平上, 品种PIERSOL VFN与其余品种之间对根结线虫的抗性均存在显著性差异,而其他12个品种间对根结线虫的抗性差异不显著。     

Activity of Hydroxamic Acids from Secale cereale Against the Plant-Parasitic Nematodes Meloidogyne incognita and Xiphinema americanum

ABSTRACT Cyclic hydroxamic acids are secondary metabolites found in the family Poaceae and have been implicated in the allelopathy of rye (Secale cereale). The toxicity of these compounds against plant-parasitic nematodes is unknown. DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one), DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one), and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) were screened in vitro against Meloidogyne incognita second-stage juveniles (J2) and eggs and mixed-stages of Xiphinema americanum. Xiphinema americanum was more sensitive to DIBOA and DIMBOA than M. incognita J2, with a maximum apparent mortality of 96 and 92% compared to 73 and 72% at 90 mug/ml. Eggs of M. incognita were less sensitive to the hydroxamic acids than J2; only DIBOA resulted in a 50% reduction in egg hatch, with a lethal concentration (LC(50)) of 74 mug/ml compared to 21 mug/ml for J2. When M. incognita J2 were exposed to DIBOA for 48 h and the compound was removed and replaced with water, the LC(50) value increased from 21.0 to 40.7 mug/ml. MBOA was not toxic to X. americanum or M. incognita eggs, but was toxic to M. incognita J2, with LC(50) values of 44 and 20 mug/ml before and after the compound was removed and replaced with water. BOA was the least toxic hydroxamic acid tested; it did not reduce M. incognita egg hatch after 1 week of exposure or increase X. americanum mortality after 24 h of exposure. While in vitro studies provide a valuable starting point in determining the toxicity of the chemical component of rye, the relevance of the data to soil remains to be determined.     

添加有机物料的微生物生防制剂对草莓灰霉病的防治

为解决微生物生防制剂在实际应用过程中防效不稳定的问题,本研究通过在不同微生物生防产品中添加有机物料,分析了添加有机物料对草莓灰霉病防治和土壤微生物数目的影响。本试验共设15个处理。微生物生防制剂（BCA）包括枯草芽胞杆菌Bacillus subtilis（Bs）,木霉菌Trichoderma（Tr）,EM菌（effective microorganisms,EM）,半量枯草芽胞杆菌+木霉菌（1/2BT）,以及无菌剂对照（CK）;有机物料用量（米糠+豆粕粉,RB）设3个水平,分别为不添加,1/2RB（15 g/m²米糠+60 g/m²豆粕粉）和RB（30 g/m²米糠+120 g/m²豆粕粉）。移栽55 d后将各处理组分均匀撒施至根周,随后覆盖地膜并滴灌保湿。结果表明,木霉菌和EM菌能够有效降低灰霉病发病率,有机物添加量一致时,微生物生防制剂的防效规律为EM > Tr > Bs > 1/2BT。木霉菌和EM菌单剂在发病较轻的试验地1中防效为60.00%~68.00%,发病严重的试验地2中防效为16.39%~29.01%。各处理中EM+RB防效最高,最高可达90.00%。添加全量米糠和豆粕粉能够显著提高生防制剂的防效。生防制剂添加有机物料的防效规律为BCA+RB > BCA+1/2RB > BCA,Tr+RB防效比Tr单剂施用提高0.17~1.1倍,EM+RB防效比EM单剂提高0.32~1.84倍。同时土壤微生物的数目随有机物料的增加而增加,1/2RB和RB可使数目分别增长0.43~8.86倍和0.93~32.72倍。以上结果表明,实际生产中施用木霉菌或EM菌能够有效防治草莓灰霉病,添加足量的有机物料可以增加土壤微生物丰度从而提高防治效果。     

Recent Advances in the Use of Plant Pathogens as Biocontrol Agents of Weeds

Abstract

Among various organisms attacking skeleton weed, Chondrilla juncea, in Europe, a rust fungus, Puccinia chondrillina, and two powdery mildews, Erysiphe cichoracearum and Leveillula taurica f. sp. chondrillae were found to be the most damaging. P. chondrillina was shown to be specific to Chondrilla and has recently been introduced into Australia where skeleton weed is important in wheat cultivation. It has already spread throughout the weed infestations and is beginning to reduce plant populations. Encouraged by the result of the Chondrilla rust, the possible use of Puccinia xanthii for the control of Xanthium strumarium and X. spinosum has also been studied. Current research on the possible use of phytopathogens in the biological control of some other weeds is reviewed.     

Biocontrol of Meloidogyne incognita on tomato using antagonistic fungi,plant‐growth‐promoting rhizobacteria and cattle manure

BACKGROUND: Biocontrol achieved by a single biocontrol agent is generally inconsistent under field conditions. The aim of the present study was to increase the competitiveness and efficacy of biocontrol agents by using them together with cattle manure. RESULTS: The effects of antagonistic fungi [Aspergillus niger v. Teigh., Paecilomyces lilacinus (Thom) Samson and Penicillium chrysogenum Thom] and plant‐growth‐promoting rhizobacteria (PGPR) [Azotobacter chroococcum Beijer., Bacillus subtilis (Ehrenberg) Cohn and Pseudomonas putida (Trev.) Mig.] were assessed with cattle manure on the growth of tomato and on the reproduction of Meloidogyne incognita (Kof. & White) Chitwood. Application of antagonistic fungi and PGPR alone and in combination with cattle manure resulted in a significant increase in the growth of nematode‐inoculated plants. The highest increase (79%) in the growth of nematode‐inoculated plants was observed when P. putida was used with cattle manure, followed by use of P. lilacinus plus cattle manure. Paecilomyces lilacinus resulted in a high reduction in galling and nematode multiplication, followed by P. putida, B. subtilis, A. niger, A. chroococcum and P. chrysogenum. The combined use of P. lilacinus with cattle manure resulted in a maximum reduction in galling and nematode multiplication. CONCLUSION: Application of P. lilacinus or P. putida with cattle manure was useful to achieve greater biocontrol of M. incognita on tomato. Copyright © 2009 Society of Chemical Industry     

An Interpretation of the Ultrastructural Response of Tomato Roots Susceptible and Resistant to Meloidogyne incognita (Kofoid et White) Chitwood

Giant cells, which are nutrient sources for the developing larvae of Meloidogyne incognita (Kofoid et White) Chitwood in susceptible tomato roots, are characterized by proliferation of cytoplasm and organelles, lobing of nuclei and irregularly thickened walls. The hypersensitive response of resistant tomato roots occurs more quickly than does the susceptible response and is characterized initially by intercellular deposits, loss of vacuolar electron-dense deposits, increased electron-density of the cytoplasm and an increase in the number of ribosomes. Eventually there is a loss of membrane-distinctness in the cell. The sequence and type of organelle changes suggest that the oesophageal secretions of the nematode into susceptible cells may de-repress a gene (s) to induce DNA synthesis. Subsequently auxin activity increases ribosomal RNA synthesis so facilitating protein synthesis and water and solute translocation. In a resistant plant the oesophageal secretions may activate a gene that produces a thermolabile enzyme that rapidly releases substances from the vacuoles or lysosomes and so manifests the hypersensitive response. The effect of this enzyme is overcome by exogenous kinetin thus supporting the role of cytokinins in the host response.     

Development of Colletotrichum gloeosporioides Restriction Enzyme-Mediated Integration Mutants as Biocontrol Agents Against Anthracnose Disease in Avocado Fruits

ABSTRACT Reduced-pathogenicity mutants of the avocado fruit pathogen Colletotrichum gloeosporioides isolate Cg-14 (teleomorph: Glomerella cingulata) were generated by insertional mutagenesis by restriction enzyme-mediated integration (REMI) transformation. Following seven transformations, 3,500 hygromycin-resistant isolates were subjected to a virulence assay by inoculation on mesocarp and pericarp of cv. Fuerte avocado fruits. Fourteen isolates showed a reduced degree of virulence relative compared with wild-type Cg-14. Two isolates, Cg-M-142 and Cg-M-1150, were further characterized. Cg-M-142 produced appressoria on avocado pericarp similar to Cg-14, but caused reduced symptom development on the fruit's pericarp and mesocarp. Isolate Cg-M-1150 did not produce appressoria; it caused much reduced maceration on the mesocarp and no symptoms on the pericarp. Southern blot analysis of Cg-M-142 and Cg-M-1150 showed REMI at different XbaI sites of the fungal genome. Pre-inoculation of avocado fruit with Cg-M-142 delayed symptom development by the wild-type isolate. Induced resistance was accompanied by an increase in the levels of preformed antifungal diene, from 760 to 1,200 mug/g fresh weight 9 days after inoculation, whereas pre-inoculation with Cg-M-1150 did not affect the level of antifungal diene, nor did it delay the appearance of decay symptoms. The results presented here show that reduced-pathogenicity isolates can be used for the biological control of anthracnose caused by C. gloeosporioides attack.     

寡糖生物防治应用及机理研究进展

植物诱导防御是当前国际生物防治学的热点内容,研究发现寡糖类物质是一类有效的诱导子,对于植物具有免疫调节作用,可以增强植物应对病虫害的能力。利用其进行植物病虫害防治是植物保护的新途径。本文将对目前应用较为广泛的寡糖诱导子来源和实际应用情况,以及寡糖诱抗机理研究中寡糖识别机制的研究进展进行综述。     

生姜对南方根结线虫侵染的生理生化反应

 本试验就生姜感染南方根结线虫后几种生理生化指标的变化进行了研究.结果表明,接种南方根结线虫后,生姜体内各种生理生化指标主要在初次侵染前期和二次侵染前期表现出一定的反应,其余时间则基本丧失这种反应,且生姜不同部位对南方根结线虫侵染的反应程度也有很大差异,以叶片的反应最为剧烈,根系次之,根茎反应最弱.结合南方根结线虫的生活周期,本文提出了生姜对南方根结线虫的生理生化反应主要表现在侵染初期,对再次侵染则部分丧失敏感性的论点.     

Host suitability and response of different vegetable genotypes to Meloidogyne incognita race 2 and Meloidogyne javanica in South Africa

The host suitability of 20 locally available genotypes of Capsicum, 10 of Daucus carota, 7 of Beta vulgaris- and 3 of Spinacea oleracea were assessed in separate greenhouse studies for resistance to Meloidogyne incognita race 2 and M. javanica, respectively. Substantial variation existed amongst the vegetable genotypes in the greenhouse screenings with regard to their host status to the respective root-knot nematode species. None of the genotypes of D. carota, B. vulgaris and S. oleracea showed resistance to the nematode species tested. However, resistance to M. incognita race 2 was identified in Capsicum genotype “Tobasco”, which was subsequently verified in a follow-up microplot trial using a range of initial population densities together with a susceptible Capsicum genotype “Paprika”. Reproduction factors of the nematodes were used as the main criterion to evaluate resistance. In the microplot trial, genotype “Tobasco” showed resistance at the lower inoculum levels but not at the higher nematode population levels. The need exists for more frequent and extensive screenings of the various vegetable genotypes in order to provide small-scale farmers with better options for improved and sustainable yields.     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.     

Population Dynamics of Biocontrol Agents and Pathogens in Soils and Rhizospheres

Understanding the dynamics between a pathogen and a biocontrol agent (BCA) in soil or in an infection court such as the rhizosphere is crucial for predicting the success of biological control. This is especially true for biological control using the strategy of reduction of initial inoculum prior to infection. By studying the population density fluxes over time, one can observe how the BCA and pathogen influence each other's population and life cycles, and how the biological and physical environment influence this relationship. Methods for quantifying fungi and bacteria in soil, including classical (dilution plating, baiting, bioassays), immunological (ELISA, IFC, immunomagnetic) and molecular (DNA hybridization, PCR, marker and reporter genes) methods, are discussed and critiqued. Finally, the value of mathematical modeling of population dynamics as a means of providing important information about the tripartite relationship between the pathogen, BCA, and host plant is reviewed.     

Differential reproduction of Meloidogyne incognita and M. javanica in watermelon cultivars and cucurbit rootstocks

The suitability of watermelon cultivars and cucurbit rootstocks as hosts to Meloidogyne incognita and M. javanica was determined in pot and field experiments. Meloidogyne incognita showed higher reproduction than did M. javanica on watermelon and cucurbit rootstocks. The watermelon cultivars did not differ in host status when challenged with these two species and supported lower nematode reproduction than the cucurbit rootstocks. Rootstocks Lagenaria siceraria cv. Pelops and Cucurbita pepo AK15 supported lower reproduction than did the squash hybrid rootstocks (C. maxima × C. moschata). Egg production increased (P < 0·05) with a rising initial inoculum level (Pi) in the non‐grafted Sugar Baby but the reproduction factor Rf (eggs per plant/Pi) was similar at two Pi levels. The total egg production in the plants grafted onto squash hybrids RS841 and Titan was greater (P < 0·05) at the higher Pi, but the Rf values were lower. The development of field‐grown non‐grafted watermelon plants was significantly stunted in plots where nematodes were detected at planting. However, no differences were observed in plots with grafted plants. In plots with nematodes, non‐grafted and Titan‐grafted plants had similar yields that were higher than that of RS841‐grafted plants. In the commercial plastic houses with grafted watermelon, the average Rf value was 42‐fold, confirming the high susceptibility of squash hybrids as rootstocks for grafted watermelon. The Titan–Sugar Baby combination was tolerant to M. javanica.     

β-氨基丁酸诱导植物抗病作用及其机理

β-氨基丁酸(BABA)作为一种结构简单的非蛋白质氨基酸,不仅具有广谱的诱导抗病活性,而且与多种化合物具有协同增效作用,是一种极具潜力的植物化学诱抗剂。综述了BABA诱导植物抗病性的特点、作用机理、使用方法及其代谢方式,并对其应用前景进行了展望。     

南方根结线虫侵染下罗汉果WRKY转录因子的鉴定分析

为了确定南方根结线虫侵染早期罗汉果植株中与侵染相关的WRKY基因,以接种南方根结线虫7 d后的罗汉果侧根为材料进行转录组测序分析。基于测序序列的Nr、SwissProt注释结果,共鉴定到21个WRKY基因,其中有4个转录因子含有2个WRKY结构域,其余17个转录因子只有1个WRKY结构域。系统发育分析表明,5个WRKY转录因子为第一家族成员,2个为第三家族成员,其余14个为第二家族成员。有8个WRKY转录因子与已报道的根结线虫诱导相关WRKY转录因子同源性较高,初步推测下调表达的SgWRKY2、SgWRKY3和SgWRKY17可能与南方根结线虫的早期侵染有关,它们很可能参与了线虫取食位点与巨细胞形成的复杂调控作用。本研究将为今后阐明根结线虫与罗汉果互作的分子机制提供参考依据。     

Production, Survival, and Evaluation of Solid-Substrate Inocula of Penicillium oxalicum, a Biocontrol Agent Against Fusarium Wilt of Tomato

ABSTRACT Production of conidia of Penicillium oxalicum (ATCC number pending), a biocontrol agent of Fusarium oxysporum f. sp. lycopersici, was tested in liquid and solid fermentation. P. oxalicum produced 250-fold more conidia in solid than in liquid fermentation at 30 days after inoculation of substrate. Solid fermentation was carried out in plastic bags (600 cm(3)) especially designed for solid fermentation (VALMIC) containing 50 g of peat/vermiculite (PV) (1:1, wt/wt) with 40% moisture, sealed, sterilized, and then inoculated with 1 ml of a conidial suspension of P. oxalicum (10(5) conidia g(-1) dry substrate), sealed again, and incubated in darkness at 20 to 25 degrees C for 30 days. Addition of amendments to PV in a proportion of 0.5 (wt/wt) significantly increased conidial production of P. oxalicum. The best production was obtained on PV plus meal of cereal grains (barley) or leguminous seeds (lentil) (100-fold higher). Conidial production obtained after 5 days of inoculation was similar to that obtained at 30 days. However, viability of conidia produced in PV plus lentil meal was 35% higher than that of conidia produced in PV plus barley meal. Changes in proportions (1:1:0.5, wt/wt/wt; 1:1:1, wt/wt/wt; 1:0.5:0.5, wt/wt/wt; 1:1:0.5, vol/vol/vol) of components of the substrate (peat/vermiculite/lentil meal) did not enhance production or viability of conidia. Optimal initial moisture in the substrate was 30 to 40%. At lower moistures, significant reductions of production of conidia were observed, particularly at 10%. There was a general decline in the number of conidia in bags with time of storage at -80, -20, 4, and 25 degrees C, or at room temperature (range from 30 to 15 degrees C), with the highest decline occurring from 60 to 180 days. Conidial viability also was reduced with time, except for conidia stored at -20 degrees C. Fresh conidia produced in solid fermentation system or those conidia stored at -20 degrees C for 180 days reduced Fusarium wilt of tomato by 49 and 61%, respectively.