Correlations between most probable number and activity of nematode-trapping fungi

ABSTRACT Soil cages were used to determine whether nematode-trapping fungi population density, as measured by most probable number (MPN) procedures, was correlated with the trapping of nematodes. Fungi studied (and trap type) were Arthrobotrys oligospora (adhesive networks), A. eudermata (adhesive networks), A. dactyloides (constricting rings), Dactylellina ellipsospora (adhesive knobs), and D. haptotyla (adhesive knobs). The fungi were formulated as assimilative hyphae in dried alginate pellets. Pellets were added to field soil, the soil was packed into 80-cm(3) cages (PVC pipe, 3.0 cm long and 3.9 cm in diameter), and the cages were buried in vineyards. After 14 to 61 days, the cages were recovered, and MPN data and trapping activity were determined. For all five fungi, MPN data were correlated with the number of pellets added. Regardless of fungus population density, A. oligospora and A. eudermata trapped few if any nematodes in soil, and consequently, trapping and fungus population density were not correlated. The correlation between population density and trapping was weak for A. dactyloides but relatively strong for D. ellipsospora and D. haptotyla. High levels of trapping by the latter two fungi required more than 10(2) fungus propagules per gram of soil.     

Augmentation of Soil with the Nematophagous Fungi Hirsutella rhossiliensis and Arthrobotrys haptotyla

In previous studies, growth of Hirsutella rhossiliensis from pelletized assimilative hyphae was reduced by other soil organisms. In the current study, sensitivity to this biotic inhibition was compared when the fungus was added to soil as pelletized hyphae or as fungus-parasitized nematodes. The hypothesis was that the natural inoculum, the parasitized nematode, would be less sensitive than the artificial inoculum, pelletized hyphae. The soil was heated to 60 degrees C for 2 h to remove putative antagonists or was not heated. The soil was packed into vials (17 cm(3)) and kept at 20 degrees C in the laboratory or packed into cages (PVC pipe sealed at the ends with 480-mum pore mesh, 80 cm(3)) and buried 22 cm deep in a vineyard. After 2 or 4 weeks, assay nematodes were added to the vials or cages (recovered from the vineyard), respectively. The assay nematodes were extracted from soil after 2.0 or 2.5 days and examined for adhesive conidia of H. rhossiliensis. Consistent with the hypothesis, H. rhossiliensis was quite sensitive to biotic inhibition when formulated as pelletized hyphae but was insensitive to biotic inhibition when formulated as parasitized nematodes. These data suggest that the activity of the H. rhossiliensis pellet could be increased if the pellet better mimicked the natural inoculum. Similar experiments with the nematode-trapping fungus Arthrobotrys haptotyla, however, exhibited an opposite trend: A. haptotyla was more sensitive to biotic inhibition when added to soil as fungus-parasitized nematodes than as pelletized hyphae. Results from laboratory and field experiments were similar.     

金龟子绿僵菌对苜蓿生长的影响及对苜蓿抗蚜性的诱导作用

为明确金龟子绿僵菌Metarhizium anisopliae在促进植物生长和提高植物抗性方面的作用,以金龟子绿僵菌拌土种植紫花苜蓿Medicago sativa,于室内测定植株的生长参数、苜蓿斑蚜Therioaphis trifolii的取食选择性及其发育历期和繁殖力。结果表明,金龟子绿僵菌处理后7 d,植株株高比对照显著增加了0.87 cm;处理后14 d,植株分枝数为3.88个,根长为54.67 mm,均显著高于对照。株龄14 d的植株接入苜蓿斑蚜,至株龄28 d时,金龟子绿僵菌处理的植株株高、分枝数和根长均较对照显著增加;同时生物量、叶绿素含量和类胡萝卜素含量也有不同程度的升高。选择性试验结果显示,24 h内选择并驻留在金龟子绿僵菌处理植株叶片上的苜蓿斑蚜数量较对照显著减少28.57%。以金龟子绿僵菌处理的植株叶片饲喂苜蓿斑蚜,1～4龄若虫的发育均有所减缓,各龄若虫的发育历期均较对照叶片饲喂的各龄若虫有所延长,而成虫寿命较以对照叶片饲喂时显著缩短了30.09%,总生命历期显著缩短了18.83%。表明金龟子绿僵菌促进了紫花苜蓿植株的生长,同时增强了植株对苜蓿斑蚜的耐害性、排趋性与...     

Disease Progress Based on Effects of Verticillium dahliae and Pratylenchus penetrans on Gas Exchange in Russet Burbank Potato

ABSTRACT The interactive effects of concomitant infection by the nematode Pratylenchus penetrans and the fungus Verticillium dahliae on symptom expression in Russet Burbank potato was studied in growth chamber experiments. Treatments were P. penetrans at three initial densities, V. dahliae at one inoculum density, the combination of the nematode at these three densities and the fungus, and a noninfested control. Gas exchange was measured nondestructively in leaf cohorts of different ages, one to three times weekly, with a LI-COR portable photosynthesis system. The single-pathogen treatments had no effect on assimilation or transpiration rates, but joint infection had a significant impact. In concomitant infection, photosynthesis was impaired more than transpiration, so estimates of leaf health were based on carbon assimilation rates only. Reductions in assimilation rate were apparent before the onset of visual symptoms. Assimilation rates decreased as much as 44% in the top, and newest, leaves of concomitantly infected plants, compared to rates in control plants. Even so, the health of newly produced leaves did not become progressively worse through time. With light use efficiency less than 0.20 mol of CO(2) fixed per mol of photosynthetically active radiation used as the criterion for disease incidence, disease progressed acropetally from the oldest to the youngest leaves. In plants infected with P. penetrans (0.8 nematodes per cm(3) of soil) in combination with V. dahliae, all leaves in cohorts 1 and 2 were symptomatic by 45 days after planting, and leaves in cohorts 3 to 6 became symptomatic at weekly intervals thereafter. For the control and single-pathogen treatments, the first time that light use efficiency fell below 0.20 in all leaves in cohort 1 was 71 days after planting. Concomitant infection reduced leaf life span by about 3 weeks. Both visual and physiological symptom expression were invariant to differences in initial nematode inoculum densities ranging from 0.8 to 2.5 nematodes per cm(3) of soil in one experiment and from 1.3 to 4.1 nematodes per cm(3) of soil in a second experiment.     

不同真菌和植物种类对苜蓿滑刃线虫繁殖的影响

为探究美国进境苜蓿滑刃线虫对真菌的取食特性以及对植物的寄生性,通过接种试验观察该线虫在链格孢、灰葡萄孢、禾谷镰刀菌、拟盘多毛孢和核盘菌等5种真菌,以及在菊花、大豆、苜蓿和三叶草等4种植物8个品种上的繁殖情况.结果表明,苜蓿滑刃线虫能取食5种真菌的菌丝体,但是在各真菌上的繁殖系数(Rf)有明显差异,在链格孢和拟盘多毛孢上...     

Phosphite Inhibits Development of the Nematodes Heterodera avenae and Meloidogyne marylandi in Cereals

ABSTRACT Phosphonic acid (H(3)PO(3)) solutions were applied to wheat or to bristle oat as soil drenches before inoculation with juveniles of the sedentary, endoparasitic nematodes Heterodera avenae or Meloidogyne marylandi. All the solutions, which were pH adjusted and added at levels as low as 0.63 mg of phosphite (HPO(3)(2-)) per plant, reduced the numbers of H. avenae females and M. marylandi egg masses. Phosphate (PO(4)(3-)), applied as potassium phosphate at the same concentrations, did not reduce the number of female nematodes on the wheat. Addition of phosphate to the phosphite solutions did not change the inhibitory effect of phosphite on H. avenae, but it reduced phosphite's effect on M. marylandi. Phosphite also reduced the number of H. avenae females when applied as many as 20 days after addition of nematodes. The phosphite treatment did not prevent M. marylandi juveniles from penetrating wheat roots or inducing giant cells. However, phosphite inhibited giant cell development: 14 days after inoculation, the giant cells in the phosphite-treated wheat were almost completely vacuolated, whereas those in untreated wheat contained dense cytoplasm.     

Species-dependent effects of border cell and root tip exudates on nematode behavior

ABSTRACT Effects of border cell and root tip exudates on root knot nematode (Meloidogyne incognita) behavior were examined. In whole-plant assays using pea, M. incognita second-stage juveniles (J2) accumulated rapidly around the 1- to 2-mm apical region ensheathed by border cells, but not in the region of elongation. Within 15 to 30 min, J2 which had accumulated within detached clumps of border cells lost motility and entered into a quiescent state. When border cells (and associated root tip exudates) were washed from pea roots prior to challenge with nematodes, no such accumulation and quiescence was induced. Attraction of nematodes by roots was species dependent: no attraction or accumulation occurred in snap bean. Using a quantitative assay, three categories of chemotaxis responses occurred: attraction (pea and alfalfa cv. Thor), repulsion (alfalfa cv. Moapa 69), and no response (snap bean and alfalfa cv. Lahonton). In contrast, total root tip exudates from all three plant species acted as a repellent for M. incognita in the sand assay. An in vitro assay was developed to characterize the induced quiescence response. When total root tip exudate from the tested legumes (as well as corn) was incubated with J2 populations, >80% of the nematodes lost motility. A similar response occurred in Caenorhabditis elegans. Border cell exudates did not induce or contribute to the induction of quiescence. Cocultivation of pea border cells with M. incognita resulted in changes in border cell shape similar to those observed in response to exogenous plant hormones. No such changes occurred in snap bean border cells. Understanding the cell- and host-specific extracellular recognition that occurs between roots and pathogenic nematodes in the early stages before infection occurs could lead to new avenues for disease control.     

Note: Effect of neem and organic amendments on nematode populations in a coastal savanna tropical soil

Nematode numbers were assessed at 2-week intervals in a coastal savanna soil amended with neem leaves and animal manure for 12 weeks under fallow conditions. The number of plant-parasitic nematodes decreased significantly with the application of the neem-based amendments, whereas the number of non-parasitic nematodes increased. http://www.phytoparasitica.org posting July 14, 2005.     

Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum

ABSTRACT A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80 degrees C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.     

Penetration, Development and Emigration of Juveniles of the Nematode Meloidogyne arenaria in Myrobalan Plum (Prunus cerasifera) Clones Bearing the Ma Resistance Genes

Penetration, development and emigration of M. arenaria in the roots of three Myrobalan plum (Prunus cerasifera) clones genetically characterized for their resistance to root-knot nematodes (RKN) were studied during the 10 (penetration) and 15 (emigration) days following the date of inoculation (D) of 2500 juveniles (J2s) per plant into the soil. Miniaturized tests were conducted on the two resistant clones P.2175 (Ma1 gene) and P.1079 (Ma2 gene) and the susceptible clone P.2032 (recessive for both genes), obtained from micropropagated plantlets and grown in mini-containers under controlled conditions at 25°C in a growth chamber. For penetration and development studies, nematodes in the roots were recovered by the acid fuchsin-lactophenol staining technique. Equivalent numbers of J2s were recovered in all the clones at D+1 and D+2. Subsequently, the numbers increased rapidly in P.2032 and were significantly different from those in P.1079 and P.2175 that remained at a low level. No swollen larvae were observed in the resistant clones. In P.2032, the first swollen larvae were observed at D+4, the first females were observed at D+12, whereas the first females with attached egg sacs and the first new-generation J2s were obtained between D+21 and D+28. Our data suggest that the resistance phenomenon does not act on the very early nematode penetration but acts later by preventing feeding-site induction and development into the third-stage. For emigration studies, plants in which J2s had been allowed to penetrate for two days (from D to D+2) were washed free of soil, repotted and then, after various periods of growth, soil-free roots were placed under a mistifier to evaluate the numbers of emigrating individuals. Emigration of J2s from the roots occured mainly from D+2 to D+4 in all the genotypes and was very limited from D+4 to D+10. There was no significant differences in the number of emigrated juveniles between the resistant and susceptible clones, indicating that emigration cannot explain the difference in the numbers of nematodes recovered in the roots.     

Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.     

Interactions of Pratylenchus thornei and Fusarium oxysporum f. sp. ciceris on Chickpea

ABSTRACT Fusarium oxysporum f. sp. ciceris and the root-lesion nematode Pratylenchus thornei coinfect chickpeas in southern Spain. The influence of root infection by P. thornei on the reaction of Fusarium wilt-susceptible (CPS 1 and PV 61) and wilt-resistant (UC 27) chickpea cultivars to F. oxysporum f. sp. ciceris race 5 was investigated under controlled and field conditions. Severity of Fusarium wilt was not modified by coinfection of chickpeas by P. thornei and F. oxysporum f. sp. ciceris, in simultaneous or sequential inoculations with the pathogens. Root infection with five nematodes per cm(3) of soil and 5,000 chlamydospores per g of soil of the fungus resulted in significantly higher numbers of propagules of F. oxysporum f. sp. ciceris with the wilt-susceptible cultivar CPS 1, but not with the wilt-resistant one. However, infection with 10 nematodes per cm(3) of soil significantly increased root infection by F. oxysporum f. sp. ciceris in both cultivars, irrespective of fungal inoculum densities (250 to 2,000 chlamydospores per g of soil). Plant growth was significantly reduced by P. thornei infection on wilt-susceptible and wilt-resistant chickpeas in controlled and field conditions, except when shorter periods of incubation (45 days after inoculation) were used under controlled conditions. Severity of root necrosis was greater in wilt-susceptible and wilt-resistant cultivars when nematodes were present in the root, irrespective of length of incubation time (45 to 90 days), densities of nematodes (5 and 10 nematodes per cm(3) of soil), fungal inocula, and experimental conditions. Nematode reproduction on the wilt-susceptible cultivars, but not on the wilt-resistant one, was significantly increased by F. oxysporum f. sp. ciceris infections under controlled and field conditions.     

Effects of three microbial broth cultures and an organic amendment on growth and populations of free living and plant-parasitic nematodes on banana

The effect of 24 treatment combinations of cultures of Streptomyces costaricanus sp. nov. (ATCC55274), Bacillus thuringiensis (ATCC55273) and a strain of Paecilomyces marquandii, nematicide (cadusaphos), and/or wheat mash on growth and response of potted banana plants (Musa AAA) and populations of Radopholus similis, Helicotylenchus multicinctus and free living nematodes were studied in Río Frío, Costa Rica. The best plant responses (height, leaf numbers, healthy root weight), lowest numbers of plant parasitic nematodes and highest numbers of free living nematodes were observed for treatments containing wheat as a component. Two treatments, viz. wheat + Streptomyces costaricanus (200-ml culture) and wheat + P. marquandii (200-ml culture), gave the overall best results. Numbers of free living nematodes increased up to 1500-fold only for treatments containing wheat. Significant positive correlations existed between numbers of free living nematodes and shoot weight, healthy root biomass, plant height, and leaf numbers. Non-wheat treatments, including nematicide only, gave the poorest responses in general. Observations of nematodes sampled 50 days following planting in wheat-containing treatments showed most of the free-living nematodes ( 90%) to be infected by nematophagous fungi (species not recorded). The results show that an organic amendment to soil, with or without a microbial component, can be an effective inducer of processes that regulate plant-parasitic nematode populations in soil.     

Differential expression of a β-1,4-endoglucanase induced by diet change in the foliar nematode Aphelenchoides fragariae

We identified and characterized a β-1,4-endoglucanase, Afr-ENG-1, in the foliar nematode Aphelenchoides fragariae that is differentially expressed when the nematode feeds on fungi or plants. When individuals from hosta plants were transferred to a fungus culture, expression of the enzyme decreased 1,812-fold after five generations on the fungus diet. Afr-eng-1 was readily detected in the genome of 75% of nematodes from the plant population but only in 38% of the diet-changed population. The gene cannot be detected in nematodes maintained on fungus for over 100 generations. Diet was also associated with changes in nematode body size and in the severity of symptoms caused on hosta leaves. Plant-diet nematodes caused larger lesions and were longer and thinner than fungus-diet nematodes. Nematodes moved from a plant diet to a fungus diet for five generations had the same body size as the nematodes that had fed on the fungus for 100 generations. Full-length sequences of Afr-eng-1 were obtained and found to encode a glycosyl hydrolase family 5 protein. This is the first β-1,4-endoglucanase and plant-parasitism-related gene described in the genus Aphelenchoides.     

Effect of inorganic phosphatic fertilizers on the efficacy of an arbuscular mycorrhiza fungus against a root-knot nematode on pyrethrum

Effects of triple super phosphate (TSP) and single super phosphate (SSP) fertilizers on efficacy of a Glomus sp. (isolate KS 14) against Meloidogyne hapla were determined. The fertilizers were applied at 150 and 300 kg ha -1 at the time of fungus inoculation. Two months later, plants were inoculated with the nematodes. Plant growth and nematode disease parameters were determined 2 months after nematode inoculation. The fertilizers at both levels improved plant growth in all treatments. In general, the fungus improved plant growth on its own or in the presence of nematodes, but not in the presence of fertilizers. Both fertilizers at both levels were more effective in improving plant growth than the fungus. The fungus showed sensitivity to inorganic P fertilizers in that the fertilizers significantly reduced fungal root colonization and its pyrethrum growth stimulative effects. The fungus suppressed nematode disease severity unlike the fertilizers. The suppressive effects of the fungus on the nematodes were in most cases reduced by the fertilizers. The nematodes, unlike the fertilizers, did not have any significant effects on root colonization by the fungus or on its ability to improve pyrethrum growth. The presence of nematodes in fertilizer or fertilizer-fungus-treated plants, however, significantly reduced pyrethrum growth.     

The effects of repeated applications of the molluscicide metaldehyde and the biocontrol nematode Phasmarhabditis hermaphrodita on molluscs, earthworms, nematodes, acarids and collembolans: a two-year study in north-west Spain

Over two years, six consecutive field experiments were done in which the chemical molluscicide metaldehyde and the nematode biocontrol agent Phasmarhabditis hermaphrodita (Schneider) were applied at the standard field rates to replicated mini-plots successively planted with lettuce, Brussels sprouts, leaf beet and cabbage, to compare the effectiveness of different treatments in reducing slug damage to the crops. Soil samples from each plot were taken prior to the start of the experiments, and then monthly, to assess the populations of slugs, snails, earthworms, nematodes, acarids and collembolans. The experiments were done on the same site and each plot received the same treatment in the six experiments. The six treatments were: (1) untreated controls, (2) metaldehyde pellets, (3 and 4) nematodes applied to the planted area 3 days prior to planting without or with previous application of cow manure slurry, (5) nematodes applied to the area surrounding the planted area 3 days prior to planting, and (6) nematodes applied to the planted area once (only in the first of the six consecutive experiments). Only the metaldehyde treatment and the nematodes applied to the planted area at the beginning of each experiment without previous application of manure significantly reduced slug damage to the plants, and only metaldehyde reduced the number of slugs contaminating the harvested plants. The numbers of slugs, snails and earthworms in soil samples were compared among the six treatments tested: with respect to the untreated controls, the numbers of Deroceras reticulatum (Müller) were significantly affected only in the metaldehyde plots, and the numbers of Arion ater L only in the plots treated with nematodes applied to the planted area 3 days prior to planting without previous application of manure; numbers of snails (Ponentina ponentina (Morelet) and Oxychilus helveticus (Blum)) were not affected by the treatment. The total numbers of all earthworm species and of Lumbricus spp were unaffected by the treatment, but Dendrobaena spp increased significantly in the plots treated with manure. The numbers of nematodes, acarids and collembolans in soil samples were compared between the untreated controls and the treatments with nematodes applied 3 days prior to planting to the planted area or to the surrounding area, without previous application of manure: the treatment had a significant effect on the number of nematodes in soil samples, but acarids and collembolans were unaffected.     

Influence of different fertilization regimes on plant-parasitic nematodes in the black soil region of Northeast China

Plant-parasitic nematodes can cause serious plant diseases and adversely affect crop production. We investigated the temporal and vertical dynamics of plant-parasitic nematodes in northeast China to determine if long-term fertilizer application suppresses the population of plant-parasitic nematodes and influences their vertical distribution in the black soil region. The long-term fertilizer application lasted for 14 years, and included three treatments: pig manure combined with chemical fertilizer (MCF), chemical fertilizer (urea and ammonium phosphate, CF), and no fertilizer (NF). A 3-year corn-wheat-soybean rotation was grown on the field site, soil samples were obtained in the corn phase and nematodes were extracted, identified and counted. Six plant-parasitic nematode genera with relative abundance over 0.1% were found in this study. Heterodera was the dominant genus in all three fertilizer treatments, and its relative abundance was highest in NF (45.4%) and lowest in MCF (32.8%). Fertilizer application had a significant effect on abundance of total soil nematodes but not on either plant-parasitic nematodes or Heterodera. Significant differences in the abundance of total soil nematodes, plant-parasitic nematodes and Heterodera were observed among soil depths and interaction of fertilizer × soil depth. Total soil nematodes were mainly distributed in the 0–20 cm soil layer, and plant-parasitic nematodes and Heterodera were mostly distributed in the 10–30 cm soil layer. Fertilizer application did not show a significant effect on plant-parasitic nematodes or Heterodera at any of the sampling depths from 0 to 80 cm. The results indicated that fertilizer has no effective control on plant-parasitic nematodes when dominant genera and their respective plant hosts exist in relatively high abundance.     

Soil pH and the Activity of a Pelletized Nematophagous Fungus

ABSTRACT When formulated as assimilative hyphae in alginate pellets, the nematophagous fungus Hirsutella rhossiliensis was more active (i.e., parasitized more assay nematodes) in an acidic vineyard soil than in a neutral vineyard soil. To determine whether soil pH explained the difference, fungus activity was measured in soil from the neutral site that had been acidified (by adding sulfuric acid) and in soil from the acidic site that had been neutralized (by adding calcium hydroxide). As hypothesized, the activity of pelletized Hirsutella rhossiliensis was negatively correlated with soil pH. Maximum activity occurred at pH(calcium chloride) 4.5, and activity gradually declined to near zero as the pH increased to 6.5 and rapidly declined to near zero as the pH dropped below 4.0. Assays performed on leached soil samples indicated that the effects of sulfuric acid and calcium hydroxide were largely due to pH rather than to specific ions or osmotic potential. The effect of pH, however, was indirect. Heating the neutral soil to 60 degrees C for 2 h did not alter soil pH or electrical conductivity but increased fungus activity to levels equivalent to those in acidified soil. We conclude that, in these two soils, heat treatment or low soil pH suppresses soil organisms that otherwise interfere with growth of Hirsutella rhossiliensis from alginate pellets.     

Isolation, Selection, and Efficacy of Pochonia chlamydosporia for Control of Rotylenchulus reniformis on Cotton

ABSTRACT The reniform nematode, Rotylenchulus reniformis, is a serious threat to cotton (Gossypium hirsutum) production in the United States, causing an annual loss of about $80 million. The objective of this study was to isolate fungi from eggs of R. reniformis and select potential biocontrol agents for R. reniformis on cotton. We focused on the fungus Pochonia chlamydosporia because it suppresses root-knot and cyst nematodes and because preliminary data indicated that it was present in Arkansas cotton fields. Soil samples were collected from six cotton fields in Jefferson County, Arkansas. A total of 117 isolates of the nematophagous fungus P. chlamydosporia were obtained. In an in vitro test, 105 of the 117 isolates parasitized fewer than 15% of R. reniformis eggs, but 12 isolates parasitized between 16 and 35% of the eggs. These 12 isolates produced from 6.8 x 10(4) to 6.9 x 10(5) chlamydospores per gram of medium in vitro, and chlamydospore production was similar on rice grain and corn grain media. In two greenhouse experiments, a single application of isolate 37 (5,000 chlamydospores per gram of soil) significantly reduced the numbers of R. reniformis on cotton roots and in soil. The three isolates (37, 26, and 14) that parasitized the most eggs in vitro were also the most effective in suppressing numbers of R. reniformis and in increasing cotton growth in the greenhouse.     

Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode

ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.