Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 10⁵ or 10⁶ colony forming units g^-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.     

Impact of salinity stress on soil-borne fungi of sugarbeet

The sugarbeet cultivar Kaumera was found to be highly susceptible to infection by the root-rot pathogens Rhizoctonia solani and Sclerotium rolfsii in the absence of salinity stress. Under this environmental condition, R. solani was more efficient than S. rolfsii in producing cell wall-degrading enzymes in infected hypocotyls. Xylanase and galactanase were most effective. The rate of cell wall degradation by R. solani was nearly 2.5 times that of S. rolfsii when cells walls of healthy hypocotyls were used as sole carbon substrate for the in vitro produced crude enzymes.Under salinity stress the pathogenicity and the performance of cell wall-degrading enzymes of R. solani and S. rolfsii varied profoundly. Pathogenicity studies showed that R. solani appeared to be more tolerant than S. rolfsii of the salinity stresses applied, and relatively more virulent to cv Kaumera. The activities of cell wall enzymes of R. solani decreased and those of S. rolfsii increased with increased salt concentration when cell wall material was used as a sole carbon source. The metabolic products produced under salinity stress by R. solani and R. solani in the cell wall amended culture media shifted the initial pH towards neutrality or slight alkalinity for R. solani and to high acidity for S. rolfsii.When model substrates were used, xyland and galactan were the most responsive substrates for degradation by the cell wall enzymes of the two fungi studied. The rate of degradation was higher for S. rolfsii than for R. solani. The excessive acidity in salt stressed S. rolfsii culture media suggested reduced activities of the enzymes involved in cell wall degradation in vivo. This may explain the decreased virulence potentialities.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Comparative Antagonistic Properties of Gliocladium virens and Trichoderma harzianum on Sclerotium rolfsii and Rhizoctonia solani—its Relevance to Understanding the Mechanisms of Biocontrol

An isolate of Trichoderma harzianum which is less effective than G. virens in suppressing S. rolfsii and R. solani was compared with G. virens for various mechanisms of antagonism in vitro, viz., antagonism in dual culture/hyphal parasitism, parasitism of sclerotia and antibiosis. G. virens and T. harzianum were equally effective in parasitizing the hyphae of R. solani. Only T. harzianum parasitized the hyphae of S. rolfsii, and the two antagonists were comparable with respect to antibiosis on the test pathogens. However, G. virens readily parasitized the sclerotia of the test pathogens and was found to be more effective than T. harzianum in destroying the sclerotia. Under SEM, G. virens was found to colonize, penetrate, and sporulate inside the sclerotia of the test pathogens.Parasitism of sclerotia is suggested as the principal mechanism of biological control of S. rolfsii and R. solani by G. virens.     

Evaluation of the leaf juice of some higher plants for their toxicity against soil borne pathogens

Out of the leaf juices of eighteen plant species screened, only Eupatorium cannabinum exhibited complete toxicity against Pythium debaryanum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii. Shade drying of the leaves had no adverse effect, while oven drying produced an adverse effect on the fungitoxicity of the leaves of E. cannabinum. The crude leaf juice of E. cannabinum successfully inhibited damping-off (Fusarium oxysporum) infection of Pisum sativum seedlings.     

Studies on a species of Monosporascus isolated from triticum

Hyphal parasitic behaviour of Fusarium oxysporum on Rhizoctonia solani and consecutive changes during this phenomenon have been investigated and studied. The hyphal parasitism was very commonly recorded between the test fungi. During the course of parasitism coiling, penetration, lysis and formation of chlamydospores by F. oxysporum on R. solani were observed. R. solani is a new host record for the mycoparasite F. oxysporum.     

Fungi associated with storage rots of cocoyams (Colocasia spp.) in Nsukka,Nigeria

Cocoyam (Colocasia spp.) corms and cormels showing spoilage symptoms were collected from many stores in Nsukka locality and examined for rot and associated fungal pathogens. Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum, and F. solani were recovered from rotten cocoyams. The representative isolates of these species caused cocoyam rot in pathogenicity tests. The rot due to A. niger, B. theobromae and C. rolfsii was extensive resulting in complete maceration of cocoyam tissue. Potassium sorbate (0.1 mg/ml) protected cocoyams from fungal rot with the exception of C. rolfsii.     

Environmental <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> Strain Cs5 Acting by Two Analogous Alkyl-Quinolones and a Didecyl-Phthalate Against a Broad Spectrum of Phytopathogens Fungi

An environmental Burkholderia cepacia strain named Cs5 was isolated and identified first using API biochemical identification system and then with 16S rDNA and recA sequence homology search. This bacterium exhibited a broad spectrum of fungicidal activities against Alternaria alternata, Aspergillus niger, Fusarium culmorum, F. graminearum, F. oxysporum and Rhizoctonia solani. In the liquid conditions, the MIC of A. niger and R. solani were reached with, respectively, 1.25–2% of the Cs5 liquid culture supernatant. However, in the solid conditions, the same inhibition was caused in the presence of 3% of the Cs5 supernatant. The exhibition of these two fungi at low concentrations of supernatant Cs5 caused various morphological changes of their mycelia which were observed by confocal microscopy. Three antifungal compounds, named Cs5-255, Cs5-257 and Cs5-446, were purified from the Cs5 culture. The structural analysis of these molecules showed that Cs5-255 and Cs5-257 are analogous and belonged to the alkyl-quinolone family, while Cs5-446 was a didecyl-phthalate, isolated for the first time from a bacterium.     

Plant growth promotion abilities and formulation of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain B 388 (MTCC6521) isolated from a temperate Himalayan location

Bacillus megaterium strain B388, isolated from rhizosphere soil of pine belonging to a temperate Himalayan location has been characterized. The plant growth promotion and biocontrol properties of the bacterium have been evaluated through petridish and broth based assays. The isolate solubilized tricalcium phosphate under in vitro conditions; maximum activity (166 μg/ml) was recorded at 28°C after 15 days of incubation. Production of indole acetic acid demonstrated in broth assays was another important plant growth promoting character. The bacterium produced diffusible and volatile compounds that inhibited the growth of two phytopathogens viz. Alternaria alternata and Fusarium oxysporum. The carrier based formulations of the bacterium resulted in increased plant growth in bioassays. The rhizosphere colonization and the viability of the cells entrapped in alginate beads were greater in comparison to coal or broth based formulations. The bacterium showed maximum similarity with Bacillus megaterium by 16S rRNA analysis.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Combination of Pseudomonas aeruginosa and Pochonia chlamydosporia for Control of Root-Infecting Fungi in Tomato

A plant growth‐promoting rhizobacterium, Pseudomonas aeruginosa strain IE‐6, and a fungal antagonist, Pochonia chlamydosporia, were tested for their ability to inhibit mycelial growth of root‐infecting fungi under laboratory conditions including Macrophomina phaseolina, Fusarium oxysporum, F. solani and Rhizoctonia solani. Biocontrol effectiveness of the bacterium and the fungus alone or in combination was also determined for the control of root‐infecting fungi under field conditions. In a dual‐culture plate assay, the colonies of P. chlamydosporia and P. aeruginosa met each other and no further growth of either organism occurred. Against M. phaseolina, F. solani and R. solani, an ethyl acetate extract of the culture filtrates of P. aeruginosa inhibited fungal growth greater than the hexane extract, but against F. oxysporum the hexane extract caused greater inhibition of fungal growth. By contrast, against M. phaseolina, F. oxysporum and F. solani, the hexane extract of P. chlamydosporia was more effective in the inhibition of fungal growth than the ethyl acetate fraction. Ethyl acetate extracts of P. aeruginosa at 1.0 mg/ml not only inhibited the radial colony growth of R. solani but also lysed the fungal mycelium. P. aeruginosa produced siderophores and hydrogen cyanide under laboratory conditions. Field experiments conducted in 1997 and repeated in 1998 revealed that Pochonia chlamydosporia and P. aeruginosa significantly suppressed the root‐infecting fungi M. phaseolina, F. oxysporum, F. solani and R. solani and that the combination of the two caused greater inhibition of the fungal pathogens than either alone. Application of P. chlamydosporia and P. aeruginosa as a soil drench also resulted in enhanced growth of tomato plants.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

Enzymes Associated with Defence against Toxic Oxygen Species in PCNB-Tolerant and Sensitive Soil Fungi

The specific activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR), which are involved in protection against toxic species of oxygen, were determined in mycelia extracts of pentachloronitrobenzene (PCNB)-tolerant and susceptible soil fungi. The organisms assayed were the highly PCNB-sensitive Rhizoctonia solani and Rhizopus arrhizus; Sclerotium rolfsii and Trichoderma harzianum, which are moderately susceptible to PCNB, and the fungicide-tolerant Fusarium oxysporum f. sp. melonis and Pythium aphanidermatum. No GPOX activity was detected in the six examined fungi. Significant differences in the specific activities of the other enzyme systems among the fungi were evident. Remarkably low levels of CAT activities were measured in R. solani. Except for T. harzianum, no meaningful differences regarding SOD, CAT and GR activities with age of the fungi cultures were observed. The electrophoretic patterns of SOD and CAT displayed dissimilarities among the fungi under study. P. aphanidermatum is more polymorphic with respect to both SOD and CAT enzyme systems as compared to the other fungi. The SOD of F. oxysporum f. sp. melonis, R. arrhizus and T. harzianum is a cuprozinc enzyme, while the mangano-SOD species was detected in S. rolfsii, R. solani and T. harzianum.     

Study of the antifungal activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> LCH001 in vitro and identification of its antifungal components

An Acinetobacter strain, given the code name LCH001 and having the potential to be an endophytic antagonist, has been isolated from healthy stems of the plant Cinnamomum camphora (L.) Presl, guided by an in vitro screening technique. The bacterium inhibited the growth of several phytopathogenic fungi such as Cryphonectria parasitica, Glomerella glycines, Phytophthora capsici, Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani. Biochemical, physiological, and 16S rDNA sequence analysis proved that it is Acinetobacter baumannii. When the filtrate from the fermentation broth of strain LCH001 was tested in vitro and in vivo, it showed strong growth inhibition against several phytopathogens including P. capsici, F. graminearum, and R. solani, indicating that suppression of the growth of the fungi was due to the presence of antifungal compounds in the culture broth. Moreover, the antifungal activity of the culture filtrate was significantly correlated with the cell growth of strain LCH001. The active metabolites in the filtrate were relatively thermally stable, but were sensitive to acidic conditions. Three antifungal compounds were isolated from the culture broth by absorption onto macropore resin, ethanol extraction, chromatography on silica gel or LH-20 columns, and crystallization. The structures of the bioactive compounds were identified by spectroscopic methods as isomers of iturin A, namely, iturin A2, iturin A3, and iturin A6. The characterization of an unusual endophytic bacterial strain LCH001 and its bioactive components may provide an alternative resource for the biocontrol of plant diseases.     

Prevalence of wilt-root rot and their associated pathogens at reproductive phase in lentil

A survey of 116 districts of nine lentil growing states covering 603 farmers' fields revealed a range of 0.7–9.3% mean plant mortality at reproductive stages in different lentil growing states of the country. The overall mean mortality was 6.3%. The main pathogens found associated with plant mortality at this stage were Fusarium oxysporum f. sp. lentis (62.0%), Rhizoctonia bataticola (25.2%) and Sclerotium rolfsii (9.8%). The minor involvement of 1.8% was that of F. solani, F. chlamydosporum. F. equisetii, and R. solani. For the first time a national scenario of lentil wilt-root rot incidence at the crucial reproductive stage and their associated pathogens is reported here.     

Decomposition of cellulose strips in relation to climate,litterfall nitrogen,phosphorus and C/N ratio in natural boreal forests

Isolates of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Penicillium sp., Rhizoctonia solani, Stemphylium sp., Thielaviopsis basicola, and Verticillium dahliae were cultured on potato–dextrose agar (PDA), barley-sand and alfalfa-sand substrates in petri-dish or in column microcosms. N-mineralization by fungi and fungal-feeding nematodes in combination or fungi alone was assessed. Numbers of Aphelenchus avenae or Aphelenchoides composticola supported by the fungi were measured every 7 days. Times for full colonization of the substrates by fungi ranged from 5 to 15 days. Rhizoctonia solani and B. cinerea on PDA supported the largest A. avenae and A. composticola populations, respectively. Penicillium sp. was a nonhost for A. composticola and A. avenae. Rhizoctonia solani, B. cinerea, V. dahliae, and F. oxysporum supported significantly more nematodes than the other four fungal species. The ranked order of fungi based on the amount of N mineralized in columns free of nematodes was A. alternata (with a rate of 0.052 μg N/g-sand per day), Stemphylium sp., V. dahliae, T. basicola, B. cinerea, F. oxysporum, R. solani, and Penicillium sp. (with a rate of 0.0045 μg N/g-sand perday). The presence of A. avenae resulted in significant increases in mineral N, compared to nematode-free columns colonized by F. oxysporum, R. solani, and T. basicola alone. The presence of A. composticola resulted in significant increases in mineral N, compared to nematode-free columns colonized by A. alternata, B. cinerea, F. oxysporum, and R. solani alone. There was more mineral N incolumns in the presence of A. composticola than A. avenae in most cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode

Antifungal antibiotic from Pseudomonas chlororaphis isolate PA23 was identified as Phenazine using TLC and HPLC. Phenazine recorded the highest inhibition zone of 21?mm with 35.55% percent inhibition of mycelial growth of Pythium aphanidermatum over control. It had a significant effect on the hyphal morphology of P. aphanidermatum and on spore germination of Botryodiplodia theobromae and Alternaria solani. Disorganization of hyphal morphology of P. aphanidermatum includes vacuolization, cell content degeneration and hyphal lysis. Similarly interaction of phenazine with Rhizoctonia solani resulted in abnormal swelling of hyphal tips was noticed in the hyphal tips. Similarly the germination of sclerotia of Macrophomina phaseolina, R. solani and Sclerotium rolfsii were completely inhibited by phenazine at a concentration 50?μl. Incubation of the eggs of the root knot nematode Meloidogyne incognita in 30?μl concentration of phenazine, completely suppressed the hatching of juveniles.     

Structure of the outer surfaces of sclerotia of certain fungi

Summary The surfaces of sclerotia of Rhizoctonia solani, Botrytis cinerea and Sclerotinia rolfsii were examined with the Stereoscan electron microscope.The periphery of the sclerotium of R. solani consists of a loose net-work of hyphae which are not sufficiently thickened to withstand the extreme desiccation that takes place when the material is coated with gold-palladium alloy.The surface of the sclerotium of B. cinerea has many closely packed hyphal tips which project outwards from the centre of the structure. The thickening of the walls of the hyphae enable them to retain their shapes. A film was observed on the most exposed areas and this may have been dried-up melanin pigment.The outer skin of the sclerotium of S. rolfsii is an almost continuous layer which is thrown into ridges and troughs to give a wavy appearance.     

Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D

Acquired resistance to the antibiotic polyoxin-D was studied in two phytopathogenic fungi, Alternaria solani Sorauer, and Sclerotium rolfsii Sacc. The ED₅₀ value of the antibiotic for A. solani was 100 μg/ml and S., rolfsii 200 μg/ml. A. solani and S. rolfsii could be trained to tolerate concentrations upto 1.000 μg/ml and 2.200 μg/ml respectively. The acquired resistance in both cases was not lost on continued subculturing in fungicide-free, media. On transfer to fungicide-free media, Polyoxinresistant strains of both the fungi showed faster growth rate and appreciable reduction in sporulation compared to the original strains. The adapted strain of A. solani showed cross-resistance to Cycloheximide and Difolatan but not to Hinosan and Bayleton.