Effects of endophytic Fusarium oxysporum towards Radopholus similis activity in absence of banana

Four endophytic fungi (Fusarium spp.) isolated from the cortical tissue of surface-sterilised banana as well as from tomato roots were tested for their capacity of biological control towards the burrowing nematode Radopholus similis on banana. The pathogenic and parasitic capacities of endophytic fungi towards R. similis were tested in in vitro experiments. No parasitism of fungi on R. similis was observed. However, nematode activity decreased significantly in the presence of all endophytic fungi in vitro when compared to nematodes in the absence of fungi. The effects of fungi on R. similis activities in the soil were tested in the absence of plants. Nematode activities were reduced significantly by 16-30% by endophytic fungi when compared to untreated soil.     

Evaluation of the resistance to two nematodes: Radopholus similis and Meloidogyne spp. in four banana genotypes in Morocco

Radopholus similis and Meloidogyne spp. are the main nematode parasites of banana plants grown under plastic shelters in Morocco. A test was made in pots to evaluate the resistance of four genotypes of banana to these nematodes. Infection by Meloidogyne spp. brought about an increase in root weight in all banana plants tested because of gall formation. The inoculation of R. similis produced a reduction in length and diameter of the pseudo-trunk as well as in root and aerial mass in all genotypes. Pisang jari buaya showed the significantly lowest number of Meloidogyne nematodes per 10 g of roots, whereas for R. similis, the significantly smallest numbers were obtained in Pisang berlin and Pisang jari buaya. Therefore, Pisang jari buaya was the only banana genotype studied to show some degree of resistance to both nematodes.     

Effect of rhizosphere fluorescent Pseudomonas strains on plant-parasitic nematodes Radopholus similis and Meloidogyne spp.

Three Pseudomonas fluorescens strains and the type strain Pseudomonas putida CFBP2066 inhibited invasion of the plant-parasitic nematodes Radopholus similis and Meloidogyne spp. in banana, maize and tomato roots. Results were, however, not always significantly different from controls. One Ps. fluorescens strain kept R. similis numbers significantly lower in banana roots after the initial invasion stage. All strains also showed an in vitro repellent effect towards the nematodes, with Meloidogyne spp. being more affected than R. similis. As Ps. putida CFBP2066 was negative for the enzymatic activities tested as well as HCN productivity, it was concluded that either other chemical bacterial compounds affected nematode infectivity or strains elicited induced systemic resistance in plants.     

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

相似穿孔线虫拮抗细菌的筛选及分子鉴定

【背景】相似穿孔线虫是一种重要的检疫性有害生物,其寄主范围广,危害具有毁灭性,已给我国农业生产造成潜在威胁。由于目前大多化学杀线剂毒性较高,筛选获得对相似穿孔线虫具有拮抗活性的微生物菌株对线虫防控具有重要意义。【方法】广泛从染病香蕉植株根际采集土壤样本,运用等比稀释涂布平板法分离菌株;采用摇瓶发酵法制备拮抗菌株上清液和发酵液。通过室内生物测定和盆栽试验,筛选出对相似穿孔线虫具有拮抗作用的细菌菌株,并通过16S rDNA的PCR扩增和序列分析,对拮抗菌株进行种类鉴定。【结果】获得5株对相似穿孔线虫具有较理想拮抗活性的细菌菌株。其中,菌株HD-86的作用效果最好,其发酵上清液处理24 h后的相似穿孔线虫死亡率达100%。盆栽试验表明,菌株HD-86发酵液处理42和70 d后对相似穿孔线虫的相对防效分别为77.34%和90.51%,均优于对照药剂阿维菌素。【结论与意义】菌株HD-86对相似穿孔线虫具有很强的拮抗作用,将其鉴定为洋葱伯克氏菌Burkholderia cepacia。本研究可为相似穿孔线虫生防制剂的研发和病害的防控提供依据。     

The development of plant-parasitic nematode infestations on micro-propagated banana plants following field control measures in Côte d'Ivoire

Of the weeds that were found in banana production areas, only Asystasia gangetica was parasitised by Radopholus similis; Helicotylenchus multicinctus and Hoplolaimus pararobustus were able to parasitise all weed species. Field trials were carried out in Cote d'Ivoire to assess the potential for using nematode-free micro-propagated banana plants following cultural and chemical methods for nematode control. Banana (Musa acuminata) cv. Poyo was examined for nematodes after weed fallow, flooding and chemical treatment. Before replanting bananas, nematode assays showed that: i) all nematode species declined but were not eliminated after a 1,3-dichloropropene soil treatment; ii) H. multicinctus, H. pararobustus and Cephalenchus emarginatus were still present after either a 2-year weed fallow (dominated by Chromolaena odorata or Asystasia gangetica) or a 10-week flooding; iii) R. similis did not persist after fallowing or flooding. All nematode species were found in plots treated with 1,3-dichloropropene and that had been planted with bullheads, suckers or nematode-free micropropagated plants. After both fallow and flooding, R. similis was reintroduced on infested planting materials (bullheads and suckers) even when they were pre-treated with a nematicide. When fallow and flooded plots were planted with nematode-free materials (vitro-plants), R. similis did not appear in the roots for two vegetative cycles. The other species, still present in the soil, invaded the roots and increased slowly in numbers. These results were confirmed in commercial banana plantations.     

Optimizing the efficacy of Paecilomyces lilacinus (strain 251) for the control of root-knot nematodes

The egg pathogenic fungus Paecilomyces lilacinus (strain 251) is a biocontrol fungus with a potential range of activity to control the worldwide most important plant parasitic nematodes. This biological nematicide may be an useful tool in an integrated approach to control mainly sedentary nematodes. Greenhouse experiments were conducted with the root-knot nematodes Meloidogyne incognita and M. hapla on tomato. P. lilacinus, formulated as WG (BIOACT WG), was incorporated into soil inoculated with root-knot nematode eggs prior to transplanting the susceptible tomato cultivar "Hellfrucht". Furthermore, soil treatments were combined with seedling treatments 24 hours before transplanting and a soil drench 2 weeks after planting, respectively. Seedling and post planting treatment was also combined with a soil treatment at planting. All single or combination treatments tested decreased the gall index and the number of egg masses compared to the untreated control 12 weeks after planting. However, the combination of the seedling treatment with a pre- or at-planting application of P. lilacinus was necessary to achieve higher levels of control. Additional post plant drenching resulted in only a slight increase In efficacy. To the feasibility of this modified application system for the control of root-knot nematodes, a yield experiment was conducted with M. hapla and the susceptible cultivar "Gnom F1 Hybrid". It could be demonstrated that the above mentioned combination of pre-planting application plus the seedling and one post plant drench gave the best control and resulted in a significant fruit yield increase in concurrence with a decrease in number of galls per root.     

Control of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum in pot trials

The common soil inhabiting nematophagous fungus Paecilomyces lilacinus (Thom) Samson and the nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were assayed for their ability to reduce the populations of three economically important plant-parasitic nematodes in pot trials. The fungi were tested individually and in combination against the root-knot nematode Meloidogyne javanica (Treub) Chitwood, cereal cyst nematode Heterodera avenae Wollenweber, or burrowing nematode Radopholus similis (Cobb) Thorne on tomato, barley and tissue cultured banana plants, respectively. In all cases, nematode populations were controlled substantially by both individual and combined applications of the fungi. Combined application of P. lilacinus and M. lysipagum reduced 62% of galls and 94% of M.␣javanica juveniles on tomato when compared to the experiment with no fungi added. Sixty five percent of H. avenae cysts were reduced on barley by combined application of fungi. Control of R. similis on banana, both in the roots and in the soil, was greatest when M. lysipagum was applied alone (86%) or in combination with P. lilacinus (96%), using a strategy where the fungi were inoculated twice in 18 weeks growth period. Overall, combined application of P. lilacinus and M. lysipagum was the most effective treatment in controlling nematode populations, although in some cases M. lysipagum alone was as effective as the combined application of fungi, particularly against M. javanica.     

A new species of catenaria parasitic on nematodes of sugarcane

Summary A new species,Catenaria vermicola, is named and described. It attacks several plant-parasitic nematodes of economic importance in sugarcane soils of Louisiana. The fungus forms numerous zoospores which encyst on the cuticle, generally near natural openings of the nematode, thereby causing infection. Within the parasitized nematodes hyphae and sporangia quickly form and destroy the victim completely. The following plant-parasitic nematodes were attacked and destroyed:Belonolaimus longicaudatus, Criconemoides rusticum, Hemicycliophora gigas, Hoplolaimus tylenchiformis, Meloidogyne larvae,Pratylenchus brachyurus, P. zeae, Radopholus similis, Tylenchulus semipenetrans larvae,Tylenchorhynchus martini, andXiphinema chambersi.     

AMF-induced biocontrol against plant parasitic nematodes in Musa sp.: a systemic effect

Although mycorrhizal colonization provides a bioprotectional effect against a broad range of soil-borne pathogens, including plant parasitic nematodes, the commercial use of arbuscular mycorrhizal fungi (AMF) as biocontrol agents is still in its infancy. One of the main reasons is the poor understanding of the modes of action. Most AMF mode of action studies focused on AMF-bacterial/fungal pathogens. Only few studies so far examined AMF-plant parasitic nematode interactions. Therefore, the aim of the study was to determine whether the AMF Glomus intraradices was able to incite systemic resistance in banana plants towards Radopholus similis and Pratylenchus coffeae, two plant parasitic nematodes using a split-root compartmental set-up. The AMF reduced both nematode species by more than 50%, even when the AMF and the plant parasitic nematodes were spatially separated. The results obtained demonstrate for the first time that AMF have the ability to induce systemic resistance against plant parasitic nematodes in a root system.     

Cryopreservation of Radopholus similis, a tropical plant-parasitic nematode

For obligate plant-parasitic nematodes, cryopreservation has advantages over the usual preservation methods on whole plants or axenic culture systems, because the latter two are labourious and time and space consuming. In addition, cross contamination among different isolates can occur easily. Moreover, specific genetic studies require maintenance of the original population. The nematode under investigation, Radopholus similis, is a plant-parasitic nematode from the humid tropics. Therefore, any treatment at low temperatures is likely to add extra stress to the nematode, making the development of a cryopreservation protocol extremely difficult. In this paper, we describe experiments to achieve a successful cryopreservation protocol for the tropical nematode R. similis using vitrification solution-based methods based on a well defined mixture of cryoprotectants in combination with ultra-rapid cooling and thawing rates. A two-step treatment was used consisting of an incubation in glycerol followed by the application of a vitrifying mixture of methanol, glycerol and glucose. After cryopreservation, the pathogenicity of the nematodes was not altered, since they could infect and reproduce on carrot discs after recovery in the Ringer solution. The cryopreservation method described can be used for routine cryopreservation of R. similis lines from different origins.     

Extraction and partial characterization of polyphenol oxidase from banana (Musa acuminata Grande naine) roots.

Polyphenol oxidase activity (PPO, EC 1.14.18.1, monophenol monooxygenase, and EC 1.10.3.2, o-diphenoloxidase) has been extensively studied in banana fruit for its role in enzymatic browning. Rapid discolouration of leaf, stem and root tissue after injury and strong pigmentation of tissue extracts indicate that PPO and phenolic compounds are ubiquitous in vegetative tissue of banana as well. They hamper biochemical and molecular studies in banana, as cumbersome adaptations of extraction protocols are required. On the other hand, PPO and phenolic compounds could be an important part of the plant's defence system against pests and diseases, including root parasitic nematodes. To facilitate future studies in this area, extraction and assay conditions for PPO from roots of banana (Musa acuminata AAA, Grande naine) were optimized. Highest enzyme activities were obtained in a 0.2 M phosphate buffer at pH 7.0 with 5% insoluble polyvinylpyrrolidone and 0.25% Triton X-100. The lowest K(m) values were obtained for dopamine and D-catechin. Monophenolase activity was shown with p-cresol. Banana root PPO was strongly inhibited by dithiothreitol and sodium metabisulfite. In root sections, oxidation of dopamine strongly co-localized with aerenchyma in the cortex. The experiments revealed indications for the involvement of root PPO and dopamine in resistance of banana against the parasitic nematode Radopholus similis.     

穿孔线虫属分类研究进展(综述)

穿孔线虫属Radopholus是一类多寄主寄生的危险性植物线虫,其中以世界性危害的相似穿孔线虫R. similis为典型代表。但穿孔线虫属的分类学研究迄今仍不完善,形态描述粗糙,分子序列匮乏。本文列出穿孔线虫属的最新种类名录,归纳了27种穿孔线虫（双卵巢类型）的主要形态分类特征,并综述该属分子亲缘进化关系研究进展,为穿孔线虫属的分类和相似穿孔线虫的准确鉴定提供形态学基础和技术支持,同时也为该属线虫分类学的研究方向提出建议。     

Comparative study on the host susceptibility of different banana cultivars to plant parasitic nematodes in Malappuram district of Kerala,India

Six cultivars of banana grown widely in Malappuram district of Kerala were evaluated for the diversity of plant parasitic nematodes in their respective rhizosphere. The genotypes evaluated include Nendran, Poovan (Mysore), Njali poovan, Mysore poovan, Chenkadali and Ponnan. The study aims on a comparative analysis of rhizosphere of different banana varieties for the occurrence of plant parasitic nematodes, diversity, frequency of occurrence and to determine the most vulnerable type of banana variety. Cobb’s decanting and sieving method was used to isolate nematodes from soil. Thirty three samples randomly collected from different blocks of Malappuram district are subjected to analysis and a ten species of nematodes were reported. Among the six varieties analysed both Nendran and Mysore Poovan variety seems to be more susceptible to phytoparasitic nematodes. With respect to eight blocks of Malappuram district, Helicotylenchus sp. shows highest prominence value followed by Radopholus similis, reniform nematode and Meloidogyne incognita.     

Purpureocillium, a new genus for the medically important Paecilomyces lilacinus

Paecilomyces lilacinus was described more than a century ago and is a commonly occurring fungus in soil. However, in the last decade this fungus has been increasingly found as the causal agent of infections in man and other vertebrates. Most cases of disease are described from patients with compromised immune systems or intraocular lens implants. In this study, we compared clinical isolates with strains isolated from soil, insects and nematodes using 18S rRNA gene, internal transcribed spacer (ITS) and partial translation elongation factor 1-α (TEF) sequences. Our data show that P. lilacinus is not related to Paecilomyces, represented by the well-known thermophilic and often pathogenic Paecilomyces variotii. The new genus name Purpureocillium is proposed for P. lilacinus and the new combination Purpureocillium lilacinum is made here. Furthermore, the examined Purpureocillium lilacinum isolated grouped in two clades based on ITS and partial TEF sequences. The ITS and TEF sequences of the Purpureocillium lilacinum isolates used for biocontrol of nematode pests are identical to those causing infections in (immunocompromised) humans. The use of high concentrations of Purpureocillium lilacinum spores for biocontrol poses a health risk in immunocompromised humans and more research is needed to determine the pathogenicity factors of Purpureocillium lilacinum.     

Effect of Endophytic Fusarium oxysporum on Host Preference of Radopholus similis to Tissue Culture Banana Plants

The burrowing nematode Radopholus similis is one of the major constraints to banana (Musa spp.) production worldwide. Resource-poor farmers can potentially manage R. similis by using naturally occurring banana endophytes, such as nonpathogenic Fusarium oxysporum, that are inoculated into tissue culture banana plantlets. At present, it is unclear at what stage in the R. similis infection process the endophytes are most effective. In this study, the effect of three endophytic F. oxysporum isolates (V5w2, Eny1.31i and Eny7.11o) on R. similis host preference of either endophyte-treated or untreated banana plants was investigated. No differences were observed between the proportion of nematodes attracted to either root segments excised from endophyte-treated or untreated plants, or in experiments using endophyte-treated and untreated tissue culture banana plantlets. These results imply that the early processes of banana plant host recognition by R. similis are not affected by endophyte infection.     

Influence of Mycorrhizal Fungus, Phosphorus,and Burrowing Nematode Interactions on Growth of Rough Lemon Citrus Seedlings

Rough lemon seedlings were grown in mycorrhizal-infested or phosphorus-amended soil (25 and 300 mg P/kg) in greenhouse experiments. Plants Were inoculated with the citrus burrowing nematode, Radopholus citrophilus (0, 50, 100, or 200 nematodes per pot). Six months later, mycorrhizal plants and nonmycorrhizal, high-P plants had larger shoot and root weights than did non-mycorrhizal, low-P plants. Burrowing nematode population densities were lower in roots of mycorrhizal or nonmycorrhizal, high-P plants than in roots of nonmycorrhizal, low-P plants; however, differences in plant growth between mycorrhizal and nonmycorrhizal plants were not significant with respect to initial nematode inoculum densities. Phosphorus content in leaf tissue was significantly greater in mycorrhizal and nonmycorrhizal, high-P plants compared with nonmycorrhizal, low-P plants. Nutrient concentrations of K, Mg, and Zn were unaffected by nematode parasitism, whereas P, Ca, Fe, and Mn were less in nematode-infected plants. Enhanced growth associated with root colonization by the mycorrhizal fungus appeared to result from improved P nutrition and not antagonism between the fungus and the nematode.     

Banana Rhizodeposition: Characterization of Root Border Cell Production and Effects on Chemotaxis and Motility of the Parasitic Nematode Radopholus similis

Rhizodeposition was collected from root tips of banana (Musa acuminata). Two varieties, Grande naine and Yangambi km5, susceptible and resistant towards the burrowing nematode Radopholus similis, respectively, were examined for root border cell production under in vitro and in soil growing conditions. Two types of cells were observed in rhizodeposition: spherical cells containing large amyloplasts, called statocytes (8% of total), and long ellipsoidal border cells (92%). Border cell production was high, related to root length and not different between the two cultivars. Rhizodeposition from plants grown in soil contained similar amounts of border cells, but viability was lower than in in vitro grown plants. Chemotaxis and motility assays were performed to test the effects of banana roots, roots without rhizodeposition, rhizodeposition, exudates and border cells on the behaviour of R. similis. Roots of both the susceptible and resistant variety attracted R. similis, but only in the presence of rhizodeposition. Isolated rhizodeposition of Yangambi km5 also attracted nematodes. Border cells and exudates did not affect nematode chemotaxis. Roots of both varieties induced quiescence in R. similis in the chemotaxis assay. Thirty to fifty percent of the nematodes became temporary quiescent in the motility assay with rhizodeposition and exudates. The effect lasted for 24 h for Grande naine and up to 3 days for Yangambi km5. Rhizodeposition collected from plants grown in soil affected neither R. similis chemotaxis nor motility. Overall, there were no indications that rhizodeposition is involved in preformed resistance against R. similis in Yangambi km5.     

Comparison of damage levels caused by Radopholus similis and Helicotylenchus multicinctus on bananas in Uganda

Field surveys were done in 1997 and 1998 in Masaka district, Uganda, an area which is experiencing a decline in banana production, to assess level of damage caused by nematodes Radopholus similis and Helicotylenchus multicinctus at farm level. Ten farms within a radius of 2 km were selected and nematode damage assessed. The major nematodes encountered were Pratylenchus goodeyi, R. similis and H. multicinctus. P. goodeyi and H. multicinctus were more abundant than R. similis but R. similis had a stronger and significant correlation with root damage. A subsequent pot trial examined pathogenicities of R. similis and H. multicinctus in pure and mixed cultures on tissue cultured‐banana plantlets. R. similis alone and in mixed population reduced root fresh weight significantly, and the percentage of root necrosis ranged between 22.8–41.6% and 18.3–45.5% for March 1998 and March 1999 trials, respectively. The difference in damage caused by R. similis alone and in mixed population was not statistically significant, and was higher than the damage caused by H. multicinctus alone. There were no significant differences in pathogenicity among the R. similis isolates from different parts of Uganda.     

Infection of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum

Studying the mode of infection of a biocontrol agent is important in order to assess its efficiency. The mode and severity of infection of nematodes by a soil saprophyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratory conditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chitwood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecilomyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphal penetration. The early developed eggs were more susceptible than the eggs containing fully developed juveniles. As observed by transmission electron microscopy, fungal hypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus also infected immature cysts of H. avenae including eggs in the cysts and the eggs of R. similis. Trapping and subsequent killing of mobile stages of nematodes by M. lysipagum were studied with the above three nematodes. In addition, plant-parasitic nematodes Pratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn) Filipjev were tested with M. lysipagum. This fungus was shown to infect mobile stages of all the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, were more susceptible to the attack than adults.