Inheritance of a one-seeded pod trait in peanut

Normally, the cultivated peanut (Arachis hypogaea L.) has predominantly 2 seeds per pod or more. Two seeds per pod are predominantly found in A. hypogaea L. subsp. hypogaea var. hypogaea (the botanical classification of the US runner and virginia market types) and in subsp. fastigiata var. vulgaris (the US spanish market type); whereas, predominantly 3 or more seeds per pod are found in subsp. fastigiata vars. fastigiata (the US valencia market type), peruviana (not marketed in the United States), and aequatoriana (not marketed in the United States), and in subsp. hypogaea var. hirsuta (not marketed in the United States). However, recently, predominantly 1 seed per pod selections were found within a Georgia cross population. Crosses involving the 1-seeded pod selection were made to determine its inheritance. The F(1), F(2), and F(3) data indicated that any 2 of 3 duplicate recessive genes designated, osp(1), osp(2), and osp(3), control the 1-seeded pod trait in peanut.     

Isolation of active DNA-binding nuclear proteins from tomato galls induced by root-knot nematodes

We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsiveLEMMI9 and 35S promoters. In noninfected tissue, the method based on nuclei enrichment through a Percoll cushion was superior for isolation of DNA-protein binding activity with both promoters. With infected roots; the method based on crude extracts performed better with theLEMMI9 promoter, whereas nuclei-enriched extracts worked better with the 35S promoter. Therefore, both methods can be used to extract proteins for DNA-binding assays from infected roots, but the method of choice may depend on the promoter under study. Both authors have contributed equally to this work.     

Limited survey on aflatoxin contamination in rice

Aflatoxins (AFS) are toxic and carcinogenic fungal metabolites. Aflatoxin B1 is the most toxic and has been classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). Samples of imported rice were analyzed for their AFS content. Finley ground rice subsamples were extracted with water/methanol (100:150 v/v) followed by purification with Immunoaffinity columns (IAC). AFS purified from extracts were determined with RP-HPLC-FLD using post column electrochemical derivatization with a Kobra Cell. Concentrations of aflatoxin B1 and total AFS in test rice samples were ≤0.123 and ≤2.58 µg/kg, respectively. Tween 80 improved recoveries (86 and 106%) of aflatoxin B1 and aflatoxin G1 from brown rice. Recoveries of Aflatoxin B2 and aflatoxin G2 were substantially reduced (non-detected to 27%) by Tween 80 used in IAC cleanup of brown rice extracts. Visible dense growth of Aspergillus parasiticus (food isolate) occurred at 25 °C but higher aflatoxin B1amounts (23.9–39.3 µg/kg) accumulated when the mold grew at 37 °C in rice seeds stored for three weeks. It could be concluded that levels of aflatoxin B1 and total AFS in rice samples were within the permissible amounts of the EU and other international legislations.     

丛枝菌根真菌对西瓜嫁接苗抗南方根结线虫病的影响

根结线虫病是西瓜主要土传病害之一,长期连作往往加重西瓜根结线虫病害。利用抗病砧木嫁接可有效减轻瓜类蔬菜线虫病害,而接种丛枝菌根真菌(arbuscular mycorrhiza fungi,AMF)则能拮抗根结线虫,提高寄主植物抗病性。本试验旨在研究嫁接技术配合接种AMF降低瓜类蔬菜病害和促进生长的效应。试验以不同抗性南瓜杂交种(Cucurbita maxima×C.moschata)"青农2号"和"青农3号"分别嫁接西瓜Citrullus lanatus品种"京欣2号","青农2号"嫁接苗和"青农3号"嫁接苗先分别接种摩西管柄囊霉Funneliformis mosseae、变形球囊霉Glomus versiforme、根内根孢囊霉Rhizophagus intraradices,后接种南方根结线虫Meloidogyne incognita,测定AMF对不同西瓜嫁接苗根结线虫病和生长的影响。结果表明,接种AMF能不同程度地减轻嫁接苗根结线虫病害,其中,以"青农2号"嫁接苗接种变形球囊霉促生防病的效应最大。这可能与AMF提高根系活力、可溶性糖含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性和过氧化氢酶(CAT)活性,降低丙二醛(MDA)含量有关。结果表明,"青农2号"嫁接苗结合接种变形球囊霉是生产上具有应用潜力的技术组合。     

真菌对黄曲霉毒素B_1污染的防治研究

黄曲霉毒素是由黄曲霉、寄生曲霉等曲霉属菌株所分泌的毒性次级代谢产物,其中,尤以黄曲霉毒素B1(Aflatoxin B1,AFB1)的毒性、致突变性、致癌性最强,而且其在农作物的栽培、收获、贮藏和加工过程中污染严重,受到全世界的广泛关注。因此,为保证食品的安全性,各国研究人员一直都在寻求安全、高效、经济、环保的方法来控制食品和饲料中AFB1的污染。近年来真菌在AFB1的生物防治方面已取得很大进展,并有部分菌株已应用于生产中。本研究就真菌对AFB1的防治机制及其前景展望进行综述。     

Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination

Summary  Aflatoxins, highly toxic and carcinogenic compounds that frequently contaminate foods and feeds, are produced by several genera in the genusAspergillus. Aspergillus flavus, the most common species causing crop contamination, is a common inhabitant of the Sonoran desert of North America where it resides in complex communities composed of diverse individuals. This diversity reflects divergent adaptation to various ecological niches. SomeA. flavus isolates that are well adapted to plant associated niches do not produce aflatoxins yet have the capacity to competitively exclude aflatoxin producers. These atoxigenic strains can serve as biological control agents for management of aflatoxins in crops. Detailed knowledge of the ecology of aflatoxin-producing fungi may lead to novel practical methods for limiting contamination. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes

A simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 10⁵spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second-stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second-generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.     

Use of pepper crop residues for the control of root-knot nematodes

The biofumigant effect of pepper crop residues (PCR) for controlling Meloidogyne incognita populations was evaluated. Under laboratory conditions, 0, 5, 10 and 20 g PCR were applied to 500 g nematode infested soil, with four replicates per treatment. After 20 days at 25 degrees C, PCR reduced significantly M. incognita populations and root galling indices in susceptible tomato cv. Marmande, and increased K, N and organic C in soil. In the field, biofumigation with PCR combined with fresh animal manures (with and without plastic cover), methyl bromide, and a control were evaluated through root galling indices on a pepper crop. Each treatment, except for the control, had a grafted and non-grafted susceptible pepper sub-treatment, with three replicates. Root galling indices were lower, and yields higher, on grafted plants, biofumigation with PCR and plastic cover, with similar values as MB treatment, suggesting that biofumigation with PCR is an efficient non-chemical alternative to control M. incognita populations, especially when applied with plastic cover, nitrogen-rich organic matter and followed by grafting on resistant pepper.     

Activity of Nemathorin,natural product and bioproducts against root-knot nematodes on tomatoes

An experimental study was carried out in pots to investigate the activity of Nemathorin, natural product and biopesticides against root-knot nematodes (RKNs) on tomatoes. Fosthiazate and abamectin proved to be the most effective treatments which suppressed the RKN population by 82.1%. Furthermore, arbuscular mycorrhizal fungus 2 (AMF2) was the superior treatment that reduced galls/root system followed by abamectin with the values of 72.5% and 67.2%, respectively. In addition, fosthiazate, cadusafos and crustacean2 gave the highest increase in the root length with the values of 55.8%, 54.6% and 54.6%, respectively. AMF2 was the most effective treatment which increases the root weight by 43.9%, while azadirachtin decreased the rootweight by 12.2% compared to untreated check. AMF2, cadusafos and crustacean2 not only increased the shoot length but also increased the shoot weight. Azadirachtin recorded the minimum increase in shoot system length and weight.     

Separation and purification of a keratinase as pesticide against root-knot nematodes

A new alkaline keratinase, which could kill Meloidogyne incognita (a root-knot nematode) was separated and purified from Bacillus sp. 50-3 in this study. The solid ammonium sulfate was selected to precipitate the enzyme and its proper adding mass was also determined. After solid ammonium sulfate precipitation and liquid chromatography on DEAE-Sephadex-A50 column, there was 17.7-fold purification with a yield of 46.5%, as determined by azokeratin as substrate. The purification effect was determined through SDS-PAGE and the molecular weight of the enzyme was found to be 27,423 Da by the MALDI-TOF-MS. When the second-stage juveniles of Meloidogyne incognita were exposed to 50 μg/ml of keratinase solution, 98.5% of Meloidogyne incognita mortality rates were obtained compared to control after 24 h. Its simple purification step and high yield from the cheap medium affords this keratinase great biotechnological potential, especially in controlling root-knot nematodes such as Meloidogyne incognita. To the best of our knowledge, this study is the first report that uses keratinase as a pesticide.     

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

? Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. ? The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. ? Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. ? The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti-feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc-IΔD86, expressed as a transgene in Arabidopsis thaliana , has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet-cyst nematode) and Meloidogyne incognita (root-knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc-IΔD86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective anti-feedant plant defence.     

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.     

Low amounts of herbivory by root-knot nematodes affect microbial community dynamics and carbon allocation in the rhizosphere

Increased carbon translocation to the rhizosphere via 'leakage' induced by low amounts of plant parasitic nematodes can foster microorganisms. The effects of the root-knot nematode Meloidogyne incognita on microbial biomass (C(mic)) and community structure (phospholipid fatty acids) in the rhizosphere of barley were studied. Inoculation densities of 2000, 4000, and 8000 nematodes were well below the threshold level for plant damage. A (13)CO(2) pulse-labelling was performed to assess the distribution of assimilated (13)C in the rhizosphere. Infection with M. incognita increased the carbon concentration in shoots, and enhanced root biomass slightly. The presence of nematodes did not affect microbial biomass, but significantly changed the allocation of the recent photosynthate. Less plant carbon was sequestered by microorganisms with increasing nematode abundance. Microbial community structure was distinctly altered in the early stages of the plant-nematode interactions. Both, bacteria and fungi, showed a positive response with 2000, and a negative one with 4000 and 8000 M. incognita added. The results suggest that low-level root herbivory still imposes a considerable carbon demand, and that proliferation of microorganisms due to increased rhizodeposition may be short-termed. The carbon flow to rhizosphere microbial communities is likely dependent on the specific nematode-plant association and the developmental stage of the nematode in the host.     

High-resolution mapping of the RMia gene for resistance to root-knot nematodes in peach

The RMia gene, which confers resistance (R) to the root-knot nematodes (RKN) Meloidogyne incognita and Meloidogyne arenaria, has been shown to segregate in the peach rootstocks Nemared, Shalil, and Juseitou on LG2 of the Prunus map. Here, we report the high-resolution mapping of RMia in Nemared, using the peach genome sequence and 790 individuals from two segregating peach populations, the F2 cross Montclar x Nemared and the four-way cross [(Pamirskij × Rubira) × (Montclar × Nemared)], in which Montclar, Pamirskij, and Rubira are susceptible (S) to RKN. Among the simple sequence repeat (SSR) markers designed for an initial flanking region of more than 1 Mb, five SSR markers specific for Nemared were characterized. The genotyping and phenotyping of recombinant individuals in this interval narrowed the gene’s location to a 300 kb physical distance between the SSR markers AMPP117 and AMPP116. In this interval, SNP polymorphisms were recovered from 1-kb-sequenced DNA fragments that were selected at 20 kb intervals. Two SNP markers (A20SNP and SNP_APP91) were shown to flank the gene in a final 92-kb region, containing four candidate genes from the TIR–NBS–LRR family. Finally, we studied the polymorphism of three closely linked markers, SNP_APP92, SNP_APP91, and AMPP117, on 28 R or S accessions from diverse Prunus species or hybrids. These markers discriminated between most R and S accessions, suggesting that at least the R sources of Nemared, Nemaguard, and Shalil share a common resistant ancestor.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.