Characterization of the <Emphasis Type="Italic">RMja</Emphasis> gene for resistance to root-knot nematodes in almond: spectrum,location, and interest for <Emphasis Type="Italic">Prunus</Emphasis> breeding

In Prunus spp., resistance genes to root-knot nematodes (RKN), Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne floridensis, confer either a complete spectrum, e.g., the Ma and Rjap genes in Myrobalan and Japanese plums (subgenus Prunophora), respectively, or a more restricted spectrum, e.g., the RMia gene (M. arenaria + M. incognita) in peach (subgenus Amygdalus). We report here characterization data of the RMja gene from the almond Alnem1, another Amygdalus source. The study of its spectrum is hampered by the inability of almond to be propagated by cuttings; we overcame this problem by using F1 and BC1 crosses with previously genotyped Myrobalan plums that conferred their rooting ability to hybrids for simultaneous evaluation to different RKN. As expected from a homozygous dominant resistance, BC1 progenies of Alnem1 segregated for resistance to M. javanica but were uniformly susceptible to M. incognita and M. floridensis, demonstrating that RMja controlled M. javanica but not M. incognita nor M. floridensis. SSR markers covering the Prunus reference map placed RMja on LG7 in the same region as Ma and Rjap and thus showed its independence from the RMia gene (LG2) of the botanically closer peach. The spectrum of this gene allows the theoretical construction of interspecific rootstocks, Myrobalan plum × (almond × peach), which cumulate RMja with Ma and RMia and are protected from each of the predominant RKN affecting Prunus, i.e., M. arenaria, M. incognita, and M. javanica, by at least two genes. This pyramiding strategy should offer to rootstock material an unprecedented guarantee of durable RKN resistance.     

<Emphasis Type="Italic">Sporothrix inflata</Emphasis>, a root-inhabiting fungus of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Q. petraea</Emphasis>

The anamorphic fungus Sporothrix inflata, known as a soil-borne fungus with worldwide distribution, was isolated for the first time from the cortex and central cylinder of living and dead roots of healthy and diseased oak trees (Quercus robur and Q. petraea). Isolation frequencies of S. inflata from oak roots varied according to the health status of trees, oak species, study sites, soil depth and root diameter. Colony morphology and growth rate of isolates are influenced by colony age and type of culture medium.     

Comparative proteomic analysis of the response of fibrous roots of nematode-resistant and -sensitive sweet potato cultivars to root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

As a major root-knot nematode (RKN), Meloidogyne incognita causes serious losses in the yield of sweet potato (Ipomoea batatas L.). To successfully colonize the host plant, RKNs elicit changes of dramatic physiological and morphological features in the plants. The expression of several genes is regulated as the nematode establishes its feeding site. Therefore, in this study, we analyzed the proteomes in the fibrous roots of sweet potato plants by an infection of RKN to understand the effect of the infection on the plant root regions. This study revealed differences in proteomes of the RKN-resistant sweet potato cultivar Juhwangmi and RKN-sensitive cultivar Yulmi. During plant growth, Juhwangmi plants were shown to be more resistant to M. incognita than Yulmi plants. No M. incognita egg formation was observed in Juhwangmi plants, whereas 587 egg masses were formed in Yulmi plants. Differentially expressed 64 spots were confirmed by proteomic analysis using 2-D gel electrophoresis with three spots up-regulated in the two cultivars during RKN infection. Of these 64 protein spots, 20 were identified as belonging to such different functional categories as the defense response, cell structure, and energy metabolism. This study provides insight into the molecular and biochemical mechanics of the defense response and metabolism of sweet potato plant during nematode invasion. We anticipate that this study will also provide a molecular basis for useful crop breeding and the development of nematode-tolerant plants.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Culturable and VBNC <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: Interactions with Chironomid Egg Masses and Their Bacterial Population

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. Recently, it was found that chironomid (nonbiting midges) egg masses serve as a reservoir for the cholera bacterium and that flying chironomid adults are possible windborne carriers of V. cholerae non-O1 non-O139. Chironomids are the most widely distributed insect in freshwater. Females deposit egg masses at the water's edge, and each egg mass contains eggs embedded in a gelatinous matrix. Hemagglutinin/protease, an extracellular enzyme of V. cholerae, was found to degrade chironomid egg masses and to prevent them from hatching. In a yearly survey, chironomid populations and the V. cholerae in their egg masses followed phenological succession and interaction of host–pathogen population dynamics. In this report, it is shown via FISH technique that most of the V. cholerae inhabiting the egg mass are in the viable but nonculturable (VBNC) state. The diversity of culturable bacteria from chironomid egg masses collected from two freshwater habitats was determined. In addition to V. cholerae, representatives of the following genera were isolated: Acinetobacter, Aeromonas, Klebsiella, Shewanella, Pseudomonas, Paracoccus, Exiguobacterium, and unidentified bacteria. Three important human pathogens, Aeromonas veronii, A. caviae, and A. hydrophila, were isolated from chironomid egg masses, indicating that chironomid egg masses may be a natural reservoir for pathogenic Aeromonas species in addition to V. cholerae. All isolates of V. cholerae were capable of degrading chironomid egg masses. This may help explain their host–pathogen relationship with chironomids. In contrast, almost none of the other bacteria that were isolated from the egg masses possessed this ability. Studying the interaction between chironomid egg masses, the bacteria inhabiting them, and V. cholerae could contribute to our understanding of the nature of the V. cholerae–egg mass interactions.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> ser. <Emphasis Type="Italic">jordanica</Emphasis> (Serotype H71), a Novel Serovariety Isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3^rd instar larvae of Drosophila melanogaster and to 2^nd stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present. Received: 9 August 2002 / Accepted: 4 September 2002     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Screening for fluorescent pseudomonades,isolated from the rhizosphere of tomato,for antagonistic activity toward <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp<Emphasis Type="Italic">. michiganensis</Emphasis>

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis, continues to be a problem for tomato growers in the Souss-Massa Draa valley, South of Morocco. Assuming that biological control is an alternative for the management of this disease, a total of 303 fluorescent pseudomonads strains isolated from roots and rhizospheric soil of tomato plants were in vitro tested against C. michiganensis subsp. michiganensis. Fluorescent pseudomonads strains which showed the highest antagonistic properties were thereafter investigated for their ability to colonize tomato roots. Our results showed that fluorescent pseudomonads are more represented in rhizospheric soils. However, the most efficient fluorescent pseudomonads isolates were found in the rhizoplane soil and the endorhizosphere. Among 42 spontaneous antibiotic resistant mutants obtained by treatment of the wild-type isolates with five antibiotics (rifampicine, nalidixic acid, ampicilline and chloramphenicol), 28 completely colonized the roots of all tomatoes seedlings used in this investigation. The 42 wild type isolates were then used for in vivo screening with the cotyledon test. Using this test, eight isolates from 42 tested induced a significant decrease of disease incidence and disease symptoms. The eight efficient isolates were then tested for their effectiveness in the protection of tomato plants in pots under greenhouse conditions. Results obtained showed that all tested isolates applied as seed and root treatments reduced significantly (P ≤ 0.001) the incidence of bacterial canker.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

Differences in interactions of aboveground and belowground herbivores on the invasive plant <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> and native host <Emphasis Type="Italic">A. sessilis</Emphasis>

Plant invasions may result in novel plant-herbivore interactions. However, we know little about whether and how invasive plants can mediate native above- and belowground herbivore interactions. In this study, we conducted greenhouse experiments to examine the interaction between a native defoliating beetle, Cassida piperata, and a native root-knot nematode, Meloidogyne incognita, on the invasive alligator weed, Alternanthera philoxeroides. We also included their native host A. sessilis in the experiments to examine whether the patterns of above- and belowground herbivore interaction vary with host plants (invasive vs. native). We analyzed total carbon and nitrogen in leaves and roots attacked by M. incognita and C. piperata. M. incognita slightly negatively affected feeding by C. piperata on A. philoxeroides, and the leaf area damaged decreased as the number of M. incognita increased. M. incognita had a negative impact on total leaf nitrogen, but had no impact on total leaf carbon. M. incognita egg production on A. philoxeroides roots decreased as the amount of damage caused by C. piperata increased. Herbivory by C. piperata did not affect total root carbon or nitrogen. M. incognita and C. piperata did not affect each other on the native plant A. sessilis. These results suggest that invasive plants can mediate native above- and belowground herbivore interactions. The knowledge of how invasive plants affect those interactions is crucial for better understanding the impacts of biological invasions on native above- and belowground organisms.     

The susceptibility of immature stages of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> to the entomopathogenic fungus <Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> on tomato and verbena foliage

Lecanicillium muscarium is a widely occurring entomopathogenic fungus. Laboratory studies were conducted to determine the efficacy of L. muscarium against different instars of Bemisia tabaci on tomato and verbena foliage after two incubation times (3 and 7 days). Significant reduction in B. tabaci numbers were recorded on fungus treated plants (p < 0.001). Second instar B. tabaci proved most susceptible to L. muscarium infection. There was no significant difference in mortality of B. tabaci second instars after either 3 or 7 days exposure to L. muscarium on either host plant. The importance of the speed of pest mortality following treatment and the potential of L. muscarium to be incorporated into an integrated pest management strategy for the biocontrol of B. tabaci on tomato and verbena plants are discussed.     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

<Emphasis Type="Italic">Azospirillum brasilense</Emphasis> siderophores with antifungal activity against <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

Anthracnose, caused by the fungus Colletotrichum acutatum is one of the most important diseases in strawberry crop. Due to environmental pollution and resistance produced by chemical fungicides, nowadays biological control is considered a good alternative for crop protection. Among biocontrol agents, there are plant growth-promoting bacteria, such as members of the genus Azospirillum. In this work, we demonstrate that under iron limiting conditions different strains of A. brasilense produce siderophores, exhibiting different yields and rates of production according to their origin. Chemical assays revealed that strains REC2 and REC3 secrete catechol type siderophores, including salicylic acid, detected by thin layer chromatography coupled with fluorescence spectroscopy and gas chromatography–mass spectrometry analysis. Siderophores produced by them showed in vitro antifungal activity against C. acutatum M11. Furthermore, this latter coincided with results obtained from phytopathological tests performed in planta, where a reduction of anthracnose symptoms on strawberry plants previously inoculated with A. brasilense was observed. These outcomes suggest that some strains of A. brasilense could act as biocontrol agent preventing anthracnose disease in strawberry.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

RAPD based genetic diversity among different isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp<Emphasis Type="Italic">. lycopersici</Emphasis> and their comparative biocontrol

Fusarium wilt of tomato (Solanum lycopersicum Mill.) caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) W. C. Snyder and H. N. Hans (Fol.), is most serious and versatile pathogen. Chemical control of disease is not satisfactory and biological control is an attractive and potential alternative to the use of chemicals to control fusarium wilt of tomato. No any bioagent is universally effective everywhere therefore, search for potential biocontrol agent is continuous process and mandatory for several and individual ecological niches. In this experiment biocontrol efficacy of five species of Aspergillus and five species of Trichoderma were evaluated in vitro against Fusarium oxysporum f. sp. lycopersici. In both the experiments (dual culture and culture filtrates) T. harzianum was found to be highly effective against the isolates of Fol. followed by A. niger biocontrol potential of A. terreus is least among all the isolates tested. Culture filtrates obtained from A. luchuensis exerted least inhibition of Fol. The most sensitive isolate of Fol. against all the antagonists tested was identified as IIVR-2 (Fol. 9). Inherent diversity among Fol. isolates, from different tomato growing regions in India, was determined using RAPD primers. The genetic similarity coefficients ranged from 0.20 to 0.96, indicating that no any two or more isolates were 100% similar. RAPD profiles revealed up to 20% genetic diversity among ten isolates of Fusarium oxysporum f. sp. lycopersici.     

24-Epibrassinolide reduces stress in nematode-infected tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants cultured <Emphasis Type="Italic">in vitro</Emphasis>

24-Epibrassinolide (EBL) is considered the most probable brassinosteroid (BR) candidate that could be used for practical application in agriculture. EBL-induced stress-protective properties were evaluated in in vitro-grown tomato (Solanum lycopersicum L.) varieties Pusa Ruby (susceptible to nematodes) and PNR-7 (resistant to nematodes) during nematode pathogenesis. Sterilized tomato seeds treated with 10^?11, 10^?9, or 10^?7 M EBL and germinated in vitro were inoculated with second-stage juveniles of Meloidogyne incognita [(Kofoid and White) Chitwood]. Whole plant analyses of morphological and biochemical parameters 7 d after inoculation showed significant improvements in plant growth and development for both varieties and a highly significant reduction in the number of galls in the susceptible variety. Increased specific activities of antioxidative enzymes (catalase, ascorbate peroxidase, glutathione reductase, glutathione peroxidase, guaiacol peroxidase, and superoxide dismutase) were observed in EBL-treated seedlings of both varieties, but increases were higher in the resistant variety. A highly significant increase in antioxidants (ascorbic acid content, total flavonoid content, total glutathione content, and total phenolic content) was observed in EBL-treated Pusa Ruby seedlings, whereas in PNR-7, significant increases were found except for total flavonoid content, which increased non-significantly. Confocal microscopic images showed amelioration of stress in roots of EBL-treated seedlings as indicated by the decrease in level of green fluorescence in them as compared to untreated and nematode-inoculated roots.     

Effects of temperature and water activity on <Emphasis Type="Italic">Lecanicillium</Emphasis> spp. conidia germination and growth,and mycosis of <Emphasis Type="Italic">Pissodes strobi</Emphasis>

Selecting entomopathogenic fungal isolates for use as biocontrol agents requires an assessment of their growth and virulence characteristics as affected by environmental conditions. Here we demonstrate a wide temperature and moisture range for colony growth, effective conidial germination and virulence against Pissodes strobi Peck (white pine weevil) of several isolates of Lecanicillium Gams and Zare, an entomopathogenic fungus distributed worldwide and indigenous to forests on Vancouver Island, British Columbia, Canada. In order to examine the potential Lecanicillium as a biological control agent, the pathogenicity of isolates collected from different geographical locations on P. strobi cadavers was assessed, and colony growth at different temperatures was evaluated. Colony growth was evident between 5 and 30°C, with optimal growth occurring at 25°C. Various combinations of water activity (0.55, 0.76, 0.85 and 0.99 a _w) and temperature (10, 15, 20, and 25°C) were also used to evaluate environmental impacts on conidial germination and cumulative mycosis of adult P. strobi. Certain Lecanicillium isolates displayed xerophilic (0.85 a _w) or psychrophilic (10°C) growth optima. Ultimately, identifying the abiotic limits of this entomopathogenic fungus will be used to determine which isolates have potential for future in situ biocontrol trials.