Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Organic acids and water-soluble phenolics produced by Paxillus sp. 60/92 together show antifungal activity against Pythium vexans under acidic culture conditions

Ectomycorrhizal fungi can produce antifungal compounds in vitro as well as in symbiosis with the host plant that can reduce root diseases. The objective of this study was to isolate antifungal compounds from culture filtrate of Paxillus sp. 60/92, which can form mycorrhizas with Picea glehnii seedlings. Culture filtrate of Paxillus sp. 60/92 showed antifungal activity against Pythium vexans at pH 3–4 but not at pH 5–10, although sterile MMN-b liquid medium (pH 3–10) did not show antifungal activity. Upon separation of antifungal compounds in the culture filtrate, antifungal activity was detected in the organic acid and water-soluble phenolics fractions adjusted to pH 3. Although antifungal activity of individual fractions was lower than that of the culture filtrate, a mixture of these fractions showed antifungal activity similar to that of the culture filtrate. Furthermore, antifungal activity of oxalic acid, which is known to be produced by Paxillus involutus, was increased by mixing with the water-soluble phenolic fraction. Our findings indicate that Paxillus sp. 60/92 produces organic acids and water-soluble phenolics that together show antifungal activity at pH 3–4 against P. vexans.     

Towards the biological control of fungal and bacterial diseases of tomato using antagonistic Streptomyces spp.

This investigation was designed to explore the potential of microbial antagonism in the control of some tomato diseases including bacterial, Fusarium and Verticillium wilts; early blight; bacterial canker. Three Streptomyces spp. were used: S. pulcher, S. canescens and S. citreofluorescens.The in vitro studies showed that an 80% concentration of the culture filtrate of either S. pulcher or S. canescens significantly inhibited spore germination, mycelial growth and spotulation of Fusarium oxysporum f.sp. lycopersici, Verticillium albo-atrum and Alternaria solani. The same concentration of filtrate of either S. pulcher or S. citreofluorescens was detrimental to the bacterial populations of Clavibacter michiganensis subsp. michiganensis and Pseudomonas solanacearum.The in vivo studies involved different treatments: soaking tomato seeds in filtrate of the antagonist prior to sowing, inoculation of the soil with the antagonist 7 days before sowing, and coating of tomato seeds with spores of the antagonist before sowing. The seed-coating treatment was the most effective in controlling all the pathogens at 42 and 63 days after sowing. Soil inoculation with the antagonist 7 days prior to sowing was less effective in controlling the tomato pathogens as compared to seed-coating. The seed-soaking treatment was the least effective in controlling the diseases concerned.The results also revealed that seed-coating with antagonistic Streptomyces spp. significantly improved tomato growth.     

Studies on some fungi isolated from the rhizosphere of tomato plants and the consequent prospect for the control of Verticillium wilt

Summary Biological control of Verticillium wilt disease with antagonistic micro-organisms was studied. Antagonism of some fungi, isolated from tomato rhizosphere, toVerticillium albo-atrum R & B. was observedin vitro. A clearly defined zone, in which the growth of the pathogen was inhibited, was observed withPenicillium spp. (includingPenicillium chrysogenum Thom) andFusarium culmorum (S.G. Sm) Sacc., whileTrichoderma viride pers. ex Fries,Gliocladium spp. andPenicillium vermiculatum Dangeard, suppressed the growth ofV. albo-atrum by penetrating, and overgrowing it. OnlyT. viride andP. vermiculatum culture filtrate added to the Dox's agar, reduced the radial growth ofV. alboatrum. Root-dip application of culture filtrates ofT. viride andP. chrysogenum was found to be most effective in controlling the disease, followed by other species ofPenicillium andGliocladium spp. WhileFusarium culmorum provided no control. Improvement of plant height and vigour with a better yield due to culture filtrate treatment occurred. Root-dip application of antagonistic fungal propagules (T. viride, P. chrysogenum) to tomato seedlings was also very effective in controlling wilt in tomato plants grown inV. albo-atrum infested soil. Dedicated to the memory of the late Prof. Ivor Isaac with whom I had the pleasure of working     

Biological control of postharvest fungal rot of yam (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.) with<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The potential of isolates of Bacillus subtilis from yam farm soil to control rot of yam in storage barns was investigated. Yam tubers inoculated in vivo with B. subtilis showed no rot while those inoculated with Aspergillus niger, Botryodiploidia theobromae or Penicillium oxalicum showed considerable rot. The set of yams in which B. subtilis and the fungi were simultaneously inoculated produced rot whereas those in which B. subtilis was inoculated a day before the fungi was inoculated were totally reduced or free of rot. Many fewer fungi were isolated from the surface of tubers treated with B. subtilis than from the untreated (control) and there was high recovery of B. subtilis (99–100%) throughout the period of storage. Rot build up was faster in uninoculated control tubers or those inoculated with a spoilage fungus, while those treated with the antagonist were totally reduced or free of rot. The culture filtrate of B. subtilis prevented spore germination in some spoilage fungi. The importance of this study in relation to farmers in developing countries is discussed.     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Trichoderma spp. tolerance to Brassica carinata seed meal for a combined use in biofumigation

Biofumigation by Brassicaceae green manure or seed meal incorporation into soil is an ecological alternative to chemical fumigation against soil-borne pathogens, based on the release of glucosinolate-derived compounds. This study aimed at investigating the tolerance of the beneficial fungus Trichoderma to these compounds in view to combined utilization with Brassica carinata seed meal (BCSM). Forty isolates of Trichoderma spp. were tested in vitro for tolerance to toxic volatiles released by BCSM and in direct contact with the meal. They were found to be generally less sensitive than the assayed pathogens (Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum), even if a fungistatic effect was observed at the highest dose (10 μmole of sinigrin). Most of them also were able to grow on BCSM and over the pathogens tested. A preliminary experiment of integrating BCSM with Trichoderma in soil was carried out under controlled conditions with the patho-system P. ultimum—sugar beet. BCSM incorporation increased pathogen population, but reduced disease incidence, probably due to indirect mechanisms. The greatest effect was achieved when BCSM was applied in combination with Trichoderma, regardless of meal ability to release isothiocyanate. These findings suggest that disease control can be improved by this integrated approach. This study also highlighted that a reduction of allyl-isothiocyanate concentration in soil could occur due to the activity of some Trichoderma isolates. This effect could protect resident or introduced Trichoderma isolates from depressing effects due to the biocidal compounds, but, on the other hand, could reduce the efficacy of biofumigation against target pathogens.     

Effect of combined inoculation ofAzospirillum brasilense and vesicular-arbuscular mycorrhiza on pearl millet (Pennisetum americanum)

Summary Growth and phosphorus uptake of pearl millet (Pennisetum americanum) on an unsterile, phosphorus-deficient soil was improved by the seed inoculation withAzospirillum brasilense or soil inoculation with the vesicular-arbuscular mycorrhizal fungi (Acaulospora,Gigaspora margarita, Glomus fasciculatum). These microorganisms acted synergistically when added simultaneously and the response was significant withAzospirillum brasilense + Gigaspora margarita andAzospirillum brasilense + Glomus fasciculatum combinations over uninoculated control as far as the dry matter content of shoots, root biomass and phosphorus uptake of the millet was concerned.     

The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria

In a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples. A pool of autoclaved bacteria was added to part of the fungus culture on the second day of fermentation to increase antibiotic production. The chloroform extract from the culture broth to which the pool of autoclaved bacteria was added showed an increase of 55%, 63% and more than 100% in activity against Staphylococcus aureus, Candida albicans and Micrococcus luteus, respectively. Also, the HPLC chromatographic profiles of the chloroform extracts from both culture conditions were different. Two active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and were identified as 3,4-dimethoxyphenol and 1,3,5-trimethoxybenzene.     

Distance effect of co-occurring tree species on pine wilt disease incidence inPinus densiflora seedlings inoculated withBursaphelenchus xylophilus

Field-grownPinus densiflora seedlings were inoculated withBursaphelenchus xylophilus and the incidence of pine wilt disease was analyzed with respect to the spatial relationships between pine seedlings and adjacent seedlings of other tree species in a pure pine stand and three stands mixed withAlnus sieboldiana, Sarothamus scoparius or naturally associated species. The disease incidence was 60.9% in a 0–40 cm distance class from the nearestA. sieboldiana seedlings and then decreased with increasing minimum distance. The mean minimum distance between pine seedlings andA. sieboldiana was significantly shorter in diseased seedlings than in healthy ones. The highest disease incidence (53.9%) was found in a 0–40 cm distance class from the nearestS. scoparius, although the relationship with minimum distance was blurred by a high incidence in the 80-cm distance class. The mean minimum distance between pine seedlings was significantly short in diseased seedlings in a pure stand, whereas it was not so short between pine seedlings and other tree species in the two stands mixed withS. scoparius and the naturally associated species. The distance effect on disease incidence was noticeable inA. sieboldiana andP. densiflora at high density. This was not so clear inS. scoparius and was not found in the naturally associated species.     

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.     

Geographic range, impact, and parasitism of lepidopteran species associated with the invasive weed Lantana camara in South Africa

Six isolates of Trichoderma were screened for antagonism to Armillaria in tea stem sections buried in the soil. The inability of Armillaria to invade Trichoderma-colonized stem sections and the reduction of its viability in the plant materials following invasion of these by Trichoderma were used as indicators of antagonism. Four isolates of the species Trichoderma harzianum significantly (P<0.001) reduced the incidence of the pathogen in the plant materials. Isolate T4 completely eliminated the pathogen from plant materials in sterile soil and also antagonized two different isolates of the pathogen in nonsterile soil. Application of this T. harzianum isolate to the soil as a wheat bran culture significantly (P<0.001) reduced viability of Armillaria in woody blocks of inoculum. Soil amendment with coffee pulp also reduced the inoculum viability but did not affect the incidence of Trichoderma in the blocks of inoculum. We conclude that the direct application of wheat bran-formulated T. harzianum into soil surrounding woody Armillaria inoculum sources can suppress the pathogen. Further, no organic amendment is needed to enhance development of the antagonist in the soil as a pre-requisite to suppressing the pathogen.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

Role of fragmentation activity in cellulose hydrolysis

Most studies of cellulose hydrolysis have been carried out on three components of the cellulolytic systems, viz, endoglucanases, exoglucanases, and cellobiases. Little attention has been paid to the fragmentation activity of certain cellulolytic systems. We have noticed that despite being a more powerful degrader of modified cellulose (CMC), the 7-day grown culture filtrate of Myrothecium verrucaria was less effective than that of Trichoderma reesei at degrading pure unmodified cellulose. Scanning electron microscopy imaging showed that one distinguishing feature of the latter is its ability to fragment (macerate) the cellulose. Cellulose particle size decreased with time as it was incubated in the culture filtrate of T. reesei at 37 °C. This was used as a pre-treatment. Pre-treated cellulose was then washed and incubated with fresh T. reesei or M. verrucaria culture filtrates. Pre-treatment increased liberation of reducing sugars during subsequent incubation of cellulose in T. reesei culture filtrate but not in subsequent incubation in M. verrucaria culture filtrate. It was hypothesized that fragmentation activity of the pre-treatment opened up attack sites for further hydrolysis, but these were not available for attack by other enzyme systems.     

Antifungal substances produced by Penicillium oxalicum strain PY-1—potential antibiotics against plant pathogenic fungi

This paper reports the isolation from soil of Penicillium strain PY-1 with strong antagonistic activity against plant pathogenic fungi. On the basis of its morphological characteristics and the sequence of the ITS region, strain PY-1 was identified as P. oxalicum. Strain PY-1 produces antifungal substances that suppress the mycelial growth of Sclerotinia sclerotiorum and many other plant pathogenic fungi tested; the highest antagonistic activity was detected at 72 h when cultured in a 250-ml flask containing 80 ml potato dextrose broth. Compared with carbendazim, the relative activity of the antifungal substances produced by strain PY-1 was approximately 4 μg active ingredient (a.i.) per milliliter. The antifungal substances were extracted with ethyl acetate and further separated by high-performance liquid chromatography (HPLC); at least two active components were discovered. The ability to control plant disease with strain PY-1 was confirmed with S. sclerotiorum, a widespread pathogenic fungus that attacks rapeseed (Brassica napus) and other plants. Spores (10⁶ or 10⁷ ml⁻¹) and filtrate (tenfold diluted or undiluted) of strain PY-1 could significantly suppress infection and/or the extent of infection by S. sclerotiorum of plants at seven-true-leaves stage. The potential of strain PY-1 for identifying new antibiotics to control fungal disease and for biological control of plant disease, for example oilseed rape stem rot, is discussed.     

Inoculated common beans are protected againstMacrophomina phaseolina byBurkholderia cepacia UPR 5C

Ashy stem blight (ASB), caused byMacrophomina phaseolina (Tassi) Goid., is a common severe disease affectingPhaseolus vulgaris in Puerto Rico and the Dominican Republic. The effect of inoculating seeds ofP. vulgaris withBurkholderia cepacia strain UPR 5C on the severity of ASB was studied under greenhouse conditions. Results of this study showed thatB. cepacia reduced the ASB disease severity by 71% and was compatible withRhizobium phaseoli CIAT 632.     

Response ofSetaria italica to inoculation withAzospirillum brasilense as compared toAzotobacter chroococcum

Summary Under controlled conditions in pots filled with sand, vermiculite and field soil, inoculation withA. brasilense-Cd ATCC 29729 or withAzotobacter chroococcum caused increases above controls in the weight and N content of panicles ofSetaria italica. In no case, however, did N increases in test plants exceed the initial total N content. High acetylene reduction activities (1,000–2,000 nmole/h/pot) could be found only in plants inoculated withAzospirillum. Inoculation withAzospirillum (strain-Cd) in the field caused a significant increase above noninoculated controls of 18.5% in shoot dry weight, ofSetaria italica. Azotobacter caused a non significant increase of 8%. No significant differences were found between yields ofSetaria italica grown in soil inoculated withA. chroococcum, and those of plants grown in the presence ofA. vinelandii. A. brasilense-Cd was more effective in the field thanA. brasilense Sp-7 ATCC 29145. The results suggest that Azospirillum may increase yields ofS. italica more efficiently than Azotobacter under local field conditions.     

Effect of culture filtrates ofXanthomonas campestris pv.pelargonii and extracts of geranium stems inoculated withX. c. pv.pelargonii on geranium callus and seedlings

Experiments were designed to determine whetherXanthomonas campestris pv.pelargonii produces a toxin which induces symptoms of bacterial blight in geranium, and is active at the cellular level. Culture filtrates ofX. c. pv.pelargonii were prepared by ethyl acetate extraction and ultrafiltration of the aqueous fraction. Culture filtrates adjusted to several pH values induced maximum disease ratings on geranium seedlings in the pH range 7–10. Geranium callus growth was significantly reduced by the filtrate in the same pH range. An active fraction could also be isolated from diseased tissue. A thin-layer chromatography-callus bioassay system detected toxin activity in the culture filtrate and in extracts of geranium stems inoculated withX. c. pv.pelargonii. Callus growth inhibition was located at R_f = 0.2–0.3 for both sources of toxin. These results suggest thatX. c. pv.pelargonii produces a toxin which causes disease symptoms, is present in diseased tissues, and inhibits callus growth. This opens the possibility of developing resistance to this pathogen by selecting cells insensitive to the toxin and regenerating plants from these cells.