In vitro evaluation of some bacterial isolates as biofertilizers and biocontrol agents against the second stage juveniles of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

Representative soil samples were collected from soil apart and the rhizosphere of different plant varieties grown in five Egyptian Governorates. The presence of plant parasitic nematodes (PPN) and free living nematodes (FLN) in the collected samples were estimated. In addition, one hundred and 65 bacterial cultures which well known as biofertilizers (some are able to fix nitrogen and the others solubilize either phosphate or potassium) were isolated. The isolated bacteria were screened depending on their rate of growth. Thirty-five cultures of fast growing nitrogen fixing bacteria (NFB), phosphate solubilizing bacteria (PSB) and potassium solubilizing bacteria (KSB) and their cultural filtrates were tested in vitro as biocontrol agents against the second stage juvenile (J_2s) of the Meloidogyne incognita. In general, higher mortality percentages of nematodes were recorded by bacterial cultures than their comparative cultural filtrates. The highest mortality percentages were recorded by cultures of NFB7, PSB2 and KSB2 (100% at dilution 1/10), while it was 99.3, 99 and 97.8% at dilution 1/100, respectively. NFB7 exhibited a high nitrogen fixation rate (4.2 μmole N₂/mL/h), while PSB2 and KSB2 effectively solubilized phosphate and potassium comparing with the control treatments (1.94 fold of available phosphate and 2.0 fold of available potassium, respectively). NFB7, PSB2 and KSB2 isolates showed the highest protease, gelatinase and chitinase activities which were thought responsible for their nematicidal effect. The three bacterial isolates were identified as Paenibacillus polymyxa, Bacillus megaterium and Bacillus circulans, respectively.     

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.     

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.     

Possibilities of the use of vinasses in the control of fungi phytopathogens

The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0 and 1, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora parasitica and 10-15% for F. oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and F. oxysporum f.sp. radicis-cucumerinum (15%). In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota.     

Free living amoebae could enhance Fusarium oxysporum growth

Free living amoebae and Fusarium oxysporum can be recovered in the same environment and may potentially interact. The presence of these protozoa could lead to an increased development of this filamentous fungus. To assess this potential risk, the interactions between two free living amoebae, Acanthamoeba castellanii and Hartmanella vermiformis, and F. oxysporum, which can be isolated from soil and water, were studied. After 48 hr of coincubation in tap water, culturable fungi were quantified. In addition, the interactions between the free living amoebae and the fungus were investigated using electron microscopy. We show that the presence of amoeba trophozoites increased the growth of F. oxysporum without fungal influence on amoebae viability. In the same way, incubation of the fungus with culture supernatants of the two amoebae induced fungal germination and increased fungal growth. The results of this study confirm that the presence of amoebae should be taken into consideration in the different environments where they may be in contact with Fusarium.     

Effect of herbizid and touchdown herbicides on soil fungi and on production of some extracellular enzymes

Glucophilic and cellulose-decomposing fungi were significantly reduced in soil samples treated with 0.019-0.152 mg a.i./kg soil of the herbicides Herbizid and Touchdown. The decrease was regularly correlated with the doses of the two herbicides and persisted till the end of the experiment (12 weeks). The isolated fungi were found to be able to produce hydrolytic extracellular enzymes in solid media but with variable capabilities. The ability to produce enzymes was adversily affected by the incorporation of herbicides in culture media. Lower doses of herbicides were occasionally promotive to enzyme production and mycelial growth of some fungi. Incorporation of 50 ppm of Herbizid and Touchdown significantly activated amylase production and mycelial dry weight in cultures of Fusarium oxysporum, Mucor hiemalis and Penicillium chrysogenum. There was a significant increase in C1-cellulase produced by F. oxysporum and P. aurantiogriseum when cultures were treated with 50, 100 and 200 ppm of Herbizid which induced also more Cx-cellulase production by P. chrysogenum. Lipase and protease production was always lower in treated than in control fungal cultures.     

Isolation and analysis of bacteria associated with spores of Gigaspora margarita

Aims:  The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita , an arbuscular mycorrhizal fungus (AMF).
Methods and Results:  First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium . In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum , Fusarium oxysporum and Rhizoctonia solani . In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum.
Conclusions:  This experiment had revealed two new bacteria species ( P. polymyxa and J. lividum ), associated with AMF spores, which functioned to suppress diseases and to solubilize P.
Significance and Impact of the Study:  AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.     

Hybridization and breeding of the benomyl resistant mutant, Trichoderma harziantum antagonized to phytopathogenic fungi by protoplast fusion

A diploid strain obtained from heterokaryons of Trichoderma harzianum by protoplast fusion grew on minimal medium containing 100ppm benomyl. This strain inhibited the growth of the phytopathogenic fungus Fusarium oxysporum f. sp. raphani on paired cultures and also protected against radish yellows and a drop in germination induced by F. oxysporum f. sp. raphani.     

The Inhibition of Fungal Growth in Resistant Cotton Plants Infected by Fusarium oxysporum f. sp. vasinfectum

The vascular colonization of cotton plants by Fusarium oxysporum f. sp. vasinfectum was determined by examining growth of the fungus from free-hand cross sections taken from 0 to six days after inoculation at various distances above the points of root inoculation. Fungal spread in both longitudinal and lateral directions in the susceptible cultivar Rowden was evident four days after inoculation, whereas fungal spread in the resistant cultivar Seabrook Sea Island was restricted. The quantity of viable fungus in infected tissues was determined from macerated tissues plated on Czapek- Dox agar. The colony counts declined within six days after inoculation in resistant Seabrook Sea Island, but not in susceptible Rowden, implying that an inhibition of fungal growth in vascular tissues occurred in resistant Seabrook Sea Island. This inhibition could contribute to the restriction of fungal spread and thus be a factor in the resistance of cotton plants to F. oxysporum f. sp. vasinfectum .     

辣椒疫病拮抗菌株筛选、鉴定及其防效

从疫病发病严重田块的健康辣椒植株根际分离到98株拮抗菌,从中筛选出两株具有广谱抗性并可在贫营养条件下生长良好的高效拮抗菌株HL-3和LZ-8.通过形态观察、生理生化特性和16S rDNA序列分析,确定HL-3为多粘类芽孢杆菌,LZ-8为短小芽孢杆菌.HL-3和LZ-8对辣椒疫霉菌丝生长抑制率分别为72%和68%.HL-3和LZ-8还对黄瓜枯萎病菌、辣椒枯萎病菌、棉花黄萎病菌、黄瓜立枯病菌、烟草黑胫病菌和番茄青枯病菌具有显著的抑制作用.盆栽试验表明,HL-3和LZ-8对辣椒苗期疫病防治效果分别为72%和83%,且对辣椒生长表现出明显的促生作用.     

The photolyase gene from the plant pathogen Fusarium oxysporum f. sp. lycopersici is induced by visible light and alpha-tomatine from tomato plant

Survival of irradiated spores from Fusarium oxysporum with ultraviolet radiation (UV) was increased following exposition to visible light, indicating that this phytopathogenic fungus has a mechanism of photoreactivation able to counteract the lethal effects of UV. A genomic sequence containing the complete photolyase gene (phr1) from F. oxysporum was isolated by heterologous hybridisation with the Neurospora crassa photolyase gene. The F. oxysporum phr1 cDNA was isolated and expressed in a photolyase deficient Escherichia coli strain. The complementation of the photoreactivation deficiency of this E. coli mutant by phr1 cDNA demonstrated that the photolyase gene from F. oxysporum encodes a functional protein. The F. oxysporum PHR1 protein has a domain characteristic of photolyases from fungi (Trichoderma harziaium, N. crassa, Magnaporthe grisea, Saccharomyces cerevisiae) to bacteria (E. coli), and clusters in the photolyases phylogenetic tree with fungal photolyases. The F. oxysporum phr1 gene was inducible by visible light. The phr1 expression was also detected in presence of alpha-tomatine, a glycoalkaloid from tomato damaging cell membranes, suggesting that phr1 is induced by this cellular stress.     

Toxigenicity of some fusaria associated with plant and human diseases in the Malaysian peninsula

In the course of a plant disease survey of the Malaysian Peninsula (Malaysia comprises the Malaysian Peninsula, Sabah and Sarawak) during the period 1981-1986, more than 1000 isolates of Fusarium were obtained from diseased plants and seeds. Two further isolates were obtained from patients admitted to hospitals in the same area. The occurrences of F. proliferatum, F. nygamai and F. longipes are new records for the Malaysian Peninsula and the association of F. solani and F. oxysporum var. redolens with human diseases does not seem to have been reported previously. Ten representative species which could be classified into seven sections of the genus were selected for studies of their toxigenicity in liquid cultures and/or on rice. Crude toxin preparations from culture filtrates or extracts of the inoculated rice were tested for toxicity to brine shrimp larvae and tobacco mesophyll protoplasts. The protoplasts were more sensitive than the brine shrimp larvae to the toxin preparations, except those from the isolates of F. solani and F. oxysporum var. redolens obtained from either humans or tobacco. The toxicity of the preparations from rice cultures per g rice was always greater than the toxicity per ml of culture filtrates from cultures grown on Czapek-Dox broth, Czapek-Dox supplemented with 1% (w/v) peptone or Czapek-Dox supplemented with 5% (w/v) tobacco extract. The activity of all toxin preparations was stable to heat. It is concluded that the occurrence of toxigenic species of Fusarium in the Malaysian Peninsula is widespread and that they may pose a serious threat to the health of human, animal and plant populations.     

Biosynthesis of inulinases by Bacillus bacteria

Biosynthesis of extracellular inulinase by bacteria Bacillus polymyxa 29, B. polymyxa 722, and B. subtilis 68 was studied. The optimal parameters for the producer growth were as follows: pH 7.0, 33-35 degrees C, the growth duration within 72 h. The presence of mineral reduced or of organic nitrogen was necessary for the enzyme biosynthesis. The inulinase biosynthesis was sharply activated in the presence of carbohydrates. B. polymyxa 722 and B. polymyxa 29 displayed the maximal activity on a starch-containing culture medium, the maximal activity of B. subtilis 68 was found in the presence of sucrose. Inulin did not induce the inulinase biosynthesis by the strains studied. The time course of bacteria growth and of the enzyme biosynthesis was studied.     

大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定

内生细菌存在于健康植物体内,一些内生细菌具有促生长、抗病和固氮等生物学功能.本项研究采用化学药剂表面灭菌方法从黑龙江省大豆品种合丰25的根、茎、叶和种子中分离到大量内生细菌,其种群数量在根部最多,为3.4×103CFU/g,在叶部次之,为2.8×103CFU/g,在茎部和种子中最少,为2.9×102 CFU/g和1.4×102CFU/g.从121株内生细菌中筛选到31株对大豆根腐病菌Fusarium oxysporum f. sp.soybean具有较强抑制作用的拮抗内生细菌,其中菌株TF28抑菌谱广,抑菌率高,对不同植物的病原菌F. oxysporum的抑菌率为80.2%～96.7%.经形态、生理生化和16S rRNA鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).     

几种真菌发酵液对致病疫霉的抑制作用

测定了８种真菌发酵液在５种不同浓度下对致病疫霉菌丝生长、游动孢子静止、静止胞萌发、附着胞形成和侵入丝形成等不同阶段的影响。结果表明,供试真菌不同浓度的发酵液,对致病疫霉上述各个阶段均有一定程度的抑制作用,并均随发酵液浓度增加,抑制作用逐渐增强,浓度为１００％时,抑制作用均达到最高。其中,立枯丝核菌发酵液的抑制作用最强,浓度为１００％时,对致病疫霉菌丝生长的抑制率达到９０．４％,而静止胞萌发率仅为２．４％,附着胞及侵入丝均未见形成。     

The role of a dark septate endophytic fungus, Veronaeopsis simplex Y34, in Fusarium disease suppression in Chinese cabbage

The soil-inhabiting fungal pathogen Fusarium oxysporum has been an increasing threat to Chinese cabbage (Brassica campestris L.). A dark septate endophytic fungus, Veronaeopsis simplex Y34, isolated from Yaku Island, Japan, was evaluated in vitro for the ability to suppress Fusarium disease. Seedlings grown in the presence of the endophyte showed a 71% reduction in Fusarium wilt disease and still had good growth. The disease control was achieved through a synergetic effect involving a mechanical resistance created by a dense network of V. simplex Y34 hyphae, which colonized the host root, and siderophore production acting indirectly to induce a resistance mechanism in the plant. Changes in the relative abundance of the fungal communities in the soil as determined by fluorescently labelled T-RFs (terminal restriction fragments), appeared 3 weeks after application of the fungus. Results showed the dominance of V. simplex Y34, which became established in the rhizosphere and out-competed F. oxysporum.     

Effect of fusaric acid and phytoanticipins on growth of rhizobacteria and Fusarium oxysporum

Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.     

Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria.

The presence of other soil microorganisms might influence the ability of rhizobacterial inoculants to promote plant growth either by reducing contact between the inoculant and the plant root or by interfering with the mechanism(s) involved in rhizobacterially mediated growth promotion. We conducted the following experiments to determine whether reductions in the extent of growth promotion of lodgepole pine mediated by Paenibacillus polymyxa occur in the presence of a forest soil isolate (Pseudomonas fluorescens M20) and whether changes in plant growth promotion mediated by P. polymyxa (i) are related to changes in P. polymyxa density in the rhizosphere or (ii) result from alterations in root hormone levels. The extent of plant growth, P. polymyxa rhizosphere density, and root hormone concentrations were determined for lodgepole pine treated with (i) a single growth-promoting rhizobacterial strain (P. polymyxa L6 or Pw-2) or (ii) a combination of bacteria: strain L6 + strain M20 or strain Pw-2 + strain M20. There was no difference in the growth of pines inoculated with strain L6 and those inoculated with strain L6 + strain M20. However, seedlings inoculated with strain Pw-2 had more lateral roots and greater root mass at 12 weeks after inoculation than plants inoculated with strain Pw-2 + strain M20. The extent of growth promotion mediated by P. polymyxa L6 and Pw-2 in each treatment was not correlated to the average population density of each strain in the rhizosphere. Bacterial species-specific effects were observed in root hormone levels: indole-3-acetic acid concentration was elevated in roots inoculated with P. polymyxa L6 or Pw-2, while dihydrozeatin riboside concentration was elevated in roots inoculated with P. fluorescens M20.