Commercial Biofungicide Research Articles

Harnessing Plant Growth–Promoting and Wilt‐Controlling Biopotential of a Consortium of Actinomycetes and Mycorrhizae in Pigeon Pea

ABSTRACTFusarium‐induced wilt significantly affects the cultivation and yield of pigeon peas. This warrants sustainable disease management while promoting plant growth. The present study investigated the biopotential of coinoculation of Streptomyces pseudogriseolus S‐9 and Rhizophagus irregularis for plant growth promotion and mitigation of the impact of Fusarium wilt on pigeon pea over three seasons at pot and field levels. Pigeon pea plants were subjected to Fusarium wilt stress and treated with different inoculation strategies, including single and combined applications of S. pseudogriseolus S‐9 and R. irregularis. Plant growth parameters and yields were assessed to evaluate the efficacy of the coinoculation. In the pot experiment, T‐6 treatment resulted in the longest root (62.56 ± 0.01 cm) and shoot (70.24 ± 0.01 cm) lengths compared to the application of commercial biofungicide T‐8 (Trichoderma). This treatment also significantly influenced the yield of potted plants. It resulted in the highest fresh root weight (62.27 ± 0.01 g), fresh shoot weight (70.24 ± 0.02 g), maximum root (55.25 ± 0.01 g) and shoot dry weights (52.25 ± 0.01 g). In the field experiment, pigeon pea plants treated with the bioinoculant also demonstrated a substantial increase (р ≤ 0.05) in total grain yield, the weight of 100 grains, and the number of filled grains compared to the control group in all experimental seasons. In vitro, antagonism assay of compatibility of mycorrhizae and bacteria showed good activity using powder formulation. Thus, the consortium application inspired the broad application of Streptomyces and Trichoderma as effective bioinoculants for wilt management and yield improvement in pigeon peas.

Efficacy of Postharvest Application of Aureobasidium pullulans to Control White Haze on Apples and Effect on the Fruit Mycobiome

White haze, an emerging disorder caused by extensive fungal growth on the apple surface, results in a compromised fruit quality and decreased marketability. The use of biological control agents could be an interesting tool to reduce its development. This work aimed to test the efficacy of two Aureobasidium pullulans strains (AP2 and PL5) in controlling white haze on stored apples. An in vivo trial was conducted by inoculating fruits with white haze causal agents (Entyloma belangeri, Golubevia pallescens, Tilletiopsis washingtonensis) and treating them with the antagonistic yeasts. Three commercial biofungicides were also included in the trial. Both A. pullulans strains, along with the Metschnikowia fructicola-based product, reduced white haze incidence after 110 days of storage at 1 ± 1 °C and after 7 days of shelf life. Furthermore, the effect of A. pullulans application on the fruit fungal microbiome was assessed. A significant impact of apple matrix and treatment on the mycobiome composition was observed. Analyses showed a good colonization of A. pullulans on the treated apples, both epiphytically and endophytically. A decrease in white haze-related fungi abundance was observed in the treated fruits. Additionally, a reduction of Ramularia spp. and modifications in the abundance of other fungal genera were detected after storage and shelf life.

Suppression of Monilinia Brown Rot by Bacillus spp. Strains

Brown rot caused by Monilinia spp. is one of the main causes of pre- and postharvest losses in stone and pome fruit production. The use of beneficial microorganisms is considered one of the most promising, safe and effective alternative methods for controlling these pathogens. This study aimed to investigate the antagonistic potential of 33 Bacillus spp. strains, in order to identify the best candidate for brown rot biocontrol. Strains identified as Bacillus amyloliquefaciens B-241 and Bacillus subtilis B-313 and B-358 were chosen for further ex situ studies on detached apple fruit. The efficacy of B-241 (87.1–93.7%) did not differ significantly from a commercially available synthetic fungicide (p > 0.05). The putative mode of action of B. amyloliquefaciens B-241 against Monilinia species is competition for nutrients and antibiosis. The ethyl acetate extract of the strain, applied at 5 and 12.5 mg/mL, was bioactive in vitro and ex situ. A HPLC analysis confirmed the presence of surfactin and bacillomycin D in the extract. However, before developing a shelf-stable product and commercial production, the spectrum and efficacy on a larger scale of the B-241 strain should be determined, and its efficacy in combination with commercial biofungicides and fungicides tested in vivo.

Potential Biocontrol Agents of Corn Tar Spot Disease Isolated from Overwintered Phyllachora maydis Stromata.

Tar spot disease in corn, caused by Phyllachora maydis, can reduce grain yield by limiting the total photosynthetic area in leaves. Stromata of P. maydis are long-term survival structures that can germinate and release spores in a gelatinous matrix in the spring, which are thought to serve as inoculum in newly planted fields. In this study, overwintered stromata in corn leaves were collected in Central Illinois, surface sterilized, and caged on water agar medium. Fungi and bacteria were collected from the surface of stromata that did not germinate and showed microbial growth. Twenty-two Alternaria isolates and three Cladosporium isolates were collected. Eighteen bacteria, most frequently Pseudomonas and Pantoea species, were also isolated. Spores of Alternaria, Cladosporium, and Gliocladium catenulatum (formulated as a commercial biofungicide) reduced the number of stromata that germinated compared to control untreated stromata. These data suggest that fungi collected from overwintered tar spot stromata can serve as biological control organisms against tar spot disease.

Mycotoxins and Other Secondary Metabolites Are Produced by Gnomoniopsis smithogilvyi When Confronted with Biological and Chemical Control Agents

Gnomoniopsis smithogilvyi (Gs) is a relevant pathogen of chestnut since it provokes significant losses worldwide. The aim of this study was to screen the effect of a new biocontrol agent (BCA) against Gs isolated from chestnut (CIMO-BCA1) on the mould’s growth as well as on the production of secondary metabolites. The chemical fungicide Horizon® (tebuconazole; HOR) and the commercial biofungicide Serenade® ASO (Bacillus amyloliquefaciens QST 713; ASO) were also tested. Three concentrations of each antifungal (HOR, ASO, and CIMO-BCA1) were faced with Gs in the growth study in a chestnut-based medium. The intermediate concentrations were used for the analyses of metabolites by LC-MS/MS. CIMO-BCA1 was also identified as B. amyloliquefaciens. All agents reduced the mould’s growth, and the CIMO-BCA1 treatment with an intermediate concentration was the most effective. The metabolite analysis revealed, for the first time, the production of two mycotoxins by Gs, including 3-nitropropionic acid and diplodiatoxin. Additionally, HOR stimulated the production of diplodiatoxin. In conclusion, Gs could present a health risk for consumers. B. amyloliquefaciens strains effectively decreased the mould’s growth, but they must be applied at effective concentrations or in combination with other strategies to completely reduce the hazard.

Comparative Performance of Fungicides and Biocontrol Products inthe Management of Fusarium Wilt of Blackberry.

Fusarium wilt of blackberry (FWB) is an emerging disease caused by a Fusarium oxysporum species complex. More than 3,000 ha of blackberry (Rubus spp.) crops have been lost in Mexico since 2011. The objectives of this research were: to evaluate the sensitivity of pathogenic F. oxysporum isolates recovered from symptomatic blackberry plants to fungicides with different modes of action; to assess the potential of these fungicides and plant resistance inducers against FWB in the greenhouse; and to determine the effects of commercial biofungicides and two indigenous strains of Trichoderma spp. on the incidence of FWB. The EC50 values of the fungicides prochloraz, thiabendazole, azoxystrobin, thiophanate-methyl, difenoconazole, triflumizole, and potassium phosphite for six pathogenic F. oxysporum isolates were determined. In a separate experiment, the fungicides acibenzolar-s-methyl (ASM), potassium phosphite, and commercial biofungicides, as well as two soil microbial inoculants and two indigenous Trichoderma strains, were tested for protection against wilt development in blackberry plants in the greenhouse. Prochloraz showed an average sensitivity for EC50 of 0.01 μg ml-1 for the tested F. oxysporum isolates, followed by difenoconazole and thiabendazole. Prochloraz and ASM proved to be the most effective treatments in the greenhouse. In contrast, potassium phosphite was ineffective in both the invitro and invivo experiments. The soil bioinoculants MicroSoil, Baktillis, T. koningiopsis, and T. asperellum significantly reduced the incidence of disease in the greenhouse. These results provide evidence for the potential of the various tools as useful components of integrated FWB management in the field.

Diversity of Powdery Mildew Mycoparasite Ampelomyces quisqualis under Natural Ecosystem and Its Molecular Characterization

The powdery mildew are the most common diseases which widely affect crops in many countries. The mildew infection appeared on leaves, petiole, buds, inflorescence and other tender tissues of crops causing 65% of the crop loss. Even though the fungicidal spray to control the disease, the residual and environmental effects are causing long term ecological imbalance in cropping system. Recently, an alternative and eco-friendly mycoparasite associated with powdery mildew pathogens was used against Erysiphe sp, Leveilulla sp, Sphaerotheca sp and Oidium sp. This study aimed to mitigated the use of the expensive and harmful fungicides to save human health and to reduce the financial costs of controlling the fungal infections of crops in the field by using a commercial biofungicide naturally associated with different species of powdery midew fungi. Our results exhibited that the isolation of the mycoparasite Ampelomyces quisqualis from the mildew pathogens was closely associated with most of the genera of powdery mildew pathogens. The natural mycoparasitization efficiency of A. quisqualis was observed and higher (81%) efficiency was recorded in Erysiphe cichoracearum followed by 76% in black gram powdery mildew caused by Erysiphe polygoni. A total of 20 mycoparasitic Ampelomyces isolates were enumerated from pycnidia of 6 different powdery mildew species that naturally infected their host plants. The pycnidial morphological variations of A.quisqualis and the largest size of pycnidia was found in the Erysiphe chichoracearum (73.54µm length × 42.15µm width) shows maximum efficiency in natural parasitization. The molecular characterization of A.quisqualis isolates based on using rDNA ITS region was carried out and sequenced. The phylogenetic analysis was performed using the Maximum likelihood technique was shown the distinct relatedness with five Ampelomyces isolates made in the present study were clustered. This is the first and detailed study on diversity of Ampelomyces and quantification of natural mycoparasitism of different genera of powdery mildew of A. quisqualis.

Seed, plant, and soil treatment with selected commercial Bacillus-based and Streptomyces-based biofungicides and chemical fungicides and development of Phytophthora blight on chile pepper

There is a need to identify combination treatments with high efficacy against Phytophthora capsici, a serious pathogen of chile pepper (Capsicum sp.) and other vegetable crops in New Mexico and worldwide. A greenhouse study was conducted to determine the effects of seed, plant, and soil treatment with selected commercial formulations of Bacillus and Streptomyces, and chemical fungicides (mefenoxam) on infection of chile pepper by P. capsici. The area under disease progress curve (AUDPC) was evaluated under seed treatment used in conjunction with soil treatment, and plant treatment used in conjunction with soil treatment. There was no significant interaction (P > 0.05) between seed treatment and soil treatment, and between plant treatment and soil treatment. Soil treatment with mefenoxam reduced AUDPC over 50% compared to control (untreated). Percent plant stand and percent plant survival were greater in soil treated with mefenoxam, Streptomyces lydicus, and S. griseoviridis than in untreated soil.

Whole Genome Sequencing Reveals Major Deletions in the Genome of M7, a Gamma Ray-Induced Mutant of Trichoderma virens That Is Repressed in Conidiation, Secondary Metabolism, and Mycoparasitism.

Trichoderma virens is a commercial biofungicide used in agriculture. We have earlier isolated a mutant of T. virens using gamma ray-induced mutagenesis. This mutant, designated as M7, is defective in morphogenesis, secondary metabolism, and mycoparasitism. The mutant does not produce conidia, and the colony is hydrophilic. M7 cannot utilize cellulose and chitin as a sole carbon source and is unable to parasitize the plant pathogens Rhizoctonia solani and Pythium aphanidermatum in confrontation assay. Several volatile (germacrenes, beta-caryophyllene, alloaromadendrene, gamma-muurolene) and non-volatile (viridin, viridiol, gliovirin, heptelidic acid) metabolites are not detected in M7. In transcriptome analysis, many genes related to secondary metabolism, carbohydrate metabolism, hydrophobicity, and transportation, among others, were found to be downregulated in the mutant. Using whole genome sequencing, we identified five deletions in the mutant genome, totaling about 250 kb (encompassing 71 predicted ORFs), which was confirmed by PCR. This study provides novel insight into genetics of morphogenesis, secondary metabolism, and mycoparasitism and eventually could lead to the identification of novel regulators of beneficial traits in plant beneficial fungi Trichoderma spp. We also suggest that this mutant can be developed as a microbial cell factory for the production of secondary metabolites and proteins.

Inhibition of Aspergillus niger by Application of Punica granatum Peels and Cucurbita maxima Seeds as Bio-fungicide

Infection on plant caused by Aspergillus niger leads to the destruction of quantity and quality of crop yields. Normally, this disease is solved by the chemical fungicides. Therefore, this study was carried out to seek a potential natural fungicide from fruit waste which is safer and economical to inhibit Aspergillus niger. Cucurbita maxima (pumpkin) seeds and Punica granatum (pomegaranate) peels were extracted using maceration method with 80 % ethanol. Brine Shrimp Lethality Assay (BLSA) was used to test the presence of bioactive components in the extracts at concentration of 10 µg/mL, 100 µg/mL and 1000 µg/mL and they are expressed in terms of LC50 (Median Lethal Concentration) respectively. The study revealed that Cucurbita maxima extract was inactive, while Punica granatum extract and the mixture of both extracts at ratio 1:1 were active at 1000 µg/mL. Furthermore, the antifungal activity of Cucurbita maxima extract, Punica granatum extract, and mixture of both extracts were further tested using well-diffusion method against A. niger at 25 mg/mL, 50 mg/mL, 75 mg/mL and 100 mg/mL respectively. The findings revealed the mixture of both extracts were exerted effectively against A. niger at the lowest concentration with 20.67±2.52 mm and this gave significant zone of inhibition. The result of the study indicates that the mixture extraction of pomegranate peels and pumpkin seeds at 25 mg/mL has a great potential to be formulated as commercial bio-fungicide.

Biological control of green mould and dry bubble diseases of cultivated mushroom (Agaricus bisporus L.) by Bacillus spp.

Compost green mould and dry bubble are the most devastating diseases of the cultivated white button mushroom. In search of efficient disease control, previously and newly isolated Bacillus spp. strains obtained from mushroom substrates were examined for antagonistic activity against Trichoderma aggressivum f. europaeum T77, Trichoderma harzianum T54 and Lecanicillium fungicola var. fungicola Ša2V6in vitro. All 33 tested Bacillus spp. strains inhibited the growth of pathogens. The strains with the strongest antagonistic activity were selected for in vivo experiments in which the efficacy of strains in pathogen suppression and their effect on mushroom yield were evaluated, in comparison with a commercial biofungicide based on Bacillus velezensis QST 713 and a fungicide based on prochloraz-manganese. In mushroom growing room trials with T. aggressivum f. europaeum, the most effective in pathogen suppression were (in decreasing order) prochloraz-Mn, Bacillus amyloliquefaciens B-241, Bacillus velezensis QST 713, Bacillus pumilus B-138 and B. subtilis B-233. The commercial prochloraz-Mn fungicide was the best at suppression of L. fungicola var. fungicola, while B. amyloliquefaciens B-241 was the most successful out of the three tested Bacillus spp. strains. In all in vivo experiments, B. amyloliquefaciens B-241 showed similar results in suppression of pathogens as the commercial biofungicide B. velezensis QST 713. Regarding the impact on mushroom productivity in trials with both pathogens, no statistically significant differences were noted when the treatments were compared with inoculated and uninoculated controls. Strain B. amyloliquefaciens B-241 showed the greatest potential for biocontrol of both compost green mould and dry bubble disease, which makes it a good candidate for further trials at commercial scale.

Biological control of rice sheath blight disease with formulation of indigenous Trichoderma strains under paddy field conditions

The effectiveness of indigenous Trichoderma strains in preventing sheath blight disease was evaluated during two growing seasons under paddy field conditions. Broom sorghum seeds were used for mass production of Trichoderma strains. Colonized seeds were ground to powder and mixed with talc and carboxymethyl cellulose. Suspensions were made from the bioformulations and sprayed onto rice plants. Effects of Trichoderma strains on disease incidence and severity as well as yield and other growth parameters were determined and compared with a chemical fungicide and a commercial biofungicide. A combined analysis of variance across two years was performed and a statistically significant effect of year, treatment and their interaction was reported. Results indicate that environmental factors and different biological fungicides had a strong effect on disease development under natural conditions. According to the results, propiconazole and T. harzianum AS12-2 resulted in the least disease severity and incidence. Overall, the efficacy of T. harzianum AS12-2 in reducing sheath blight development was significantly better than other Trichoderma treatments and was comparable to the conventional fungicide.

Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein.

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline-glycine-tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.

Integration of biological and chemical control of brown rot of stone fruits to reduce disease incidence on fruits and minimize fungicide residues in juice

Brown rot is a major disease of stone fruits caused by Monilinia spp. It can reach high incidence that leads to considerable losses during storage. Our work aimed at the evaluation of innovative biological and integrated approaches to control brown rot of apricots and peaches, and of their influence on the presence of fungicide residues both in the harvested fruits and derived peach juices. In this work, we report the results of a two-year field investigation carried out in 5 different farms in Southern Italy. Plant health management strategy was based on two treatments performed in the last thirty days before harvest. Two biocontrol agents (BCAs), the yeast Rhodotorula kratochvilovae strain LS11 and the commercial biofungicide Serenade Max® (based on Bacillus subtilis strain QST 713), were applied alone (biocontrol) or in combination with low doses of the fungicides (25% of full dosage, integrated control) cyprodinil (30 days before harvest), and boscalid (7 days before harvest). The highest protection was recorded in conventional (fungicides applied at full dosage) and integrated control treatments, both on apricots and peaches. In the integrated control, residues of fungicides in fruits and in peach juice were significantly lower compared to those of the conventional control in both fruit species. Our data show that biological and integrated control based on two key treatments at thirty and seven days before harvest can be considered as effective and safer control strategies of brown rot of stone fruits. Furthermore, the applied strategies can dramatically reduce and even set to zero the level of fungicide residues in derived fruit juices.

Mechanisms of action of three isolates of Pseudomonas fluorescens active against postharvest grey mold decay of apple during commercial storage

The antagonistic activity of three isolates of Pseudomonas fluorescens, 1–112, 2–28 and 4–6, to control Botrytis cinerea (grey mold) under commercial cold storage conditions with two varieties of apple, ‘Ambrosia’ and ‘Spartan’, and possible mechanisms involved were investigated. All three isolates of P. fluorescens significantly reduced the size of the lesion and the incidence of grey mold on ‘Ambrosia’ apples after 15weeks in cold storage, in comparison to control fruits, and disease control was comparable to that of the commercial biofungicide Bio-Save® (Pseudomonas syringae). On ‘Spartan’ apples only isolates 1–112 and 4–6 significantly reduced the size of the lesion and the incidence of grey mold, after 15weeks in cold storage, and both isolates were comparable to the synthetic fungicide Scholar® (fludioxonil). Living cells of P. fluorescens or their metabolites markedly inhibited spore germination of B. cinerea in filter-sterilized apple juice (FSAJ). Cell-free supernatant and volatile organic compounds (VOCs) produced in vitro by P. fluorescens inhibited the mycelial growth of B. cinerea by 58.9 and 82.2%, respectively. Scanning electron microscopy (SEM) observations indicated that P. fluorescens colonized the wounds of apples and isolates 1–112 and 4–6 formed a biofilm at the wound site. Competition for nutrients and space, production of toxic metabolites and biofilm formation may play a role in the antagonism of P. fluorescens 1–112, 2–28 and 4–6 to B. cinerea.

Tunisian isolates of Trichoderma spp. and Bacillus subtilis can control Botrytis fabae on faba bean

ABSTRACTFaba bean crops worldwide are often attacked by different diseases, particularly chocolate spot caused by Botrytis fabae Sard. Fungal and bacterial isolates collected from faba bean and barley leaves in Tunisia were evaluated for their antagonistic potential against B. fabae. In a test on detached leaves, the highest rate of decrease in disease severity was scored by Trichoderma viride, followed by T. harzianum, the fungicide Carbendazim then Bacillus subtilis. Under glasshouse conditions, all tested fungi resulted in significant disease severity reduction. T. viride reduced the rate of chocolate spot infestation on leaves and stems by 35% and 31.5%, respectively, when the rate on the control was 100%. For T. harzianum, Carbendazim and B. Subtilis, the rates of infestation on the leaves were 41.7%, 43.1% and 59.7%, respectively. On the stems, T. harzianum scored the lowest rate of 54.2% followed by B. subtilis with 79.2% then Carbendazim with 87.5%. Two consecutive seasons of field trials using the Trichoderma species, B. subtilis and Carbendazim showed significant and consistent reduction in the severity of chocolate spot infestation rates. The highest protection against the disease was obtained from T. viride. Based on these results, Tunisian isolates of Trichoderma spp. can be recommended for developing commercial bio-fungicides for integrated management of chocolate spot.

Biological control of cotton seedling diseases by fluorescent pseudomonas spp

Seedling root rot seen in many plants including cotton is an important disease that leads to large economic losses. Human health and the environment are negatively affected as a result of using fungicides for disease control. The goal of this study was to determine the effects of fluorescent Pseudomonas (FP) bacteria against seedling root rot pathogens both in vitro and in vivo conditions. 59 FP isolates obtained from the rhizosphere of cotton and weeds on the field were tested by dual-culture assays in vitro. After applying effective FP isolates on the seeds, antagonistic effects against the seedling root rot pathogens were investigated in a climate chamber. Resulting of dual-culture tests, FP40 had maximum effect (49.60%) against Rhizoctonia solani. Besides, FP51, FP48 and FP35 had highest impact as 43.80%, 43.50%, and 43.10% against Fusarium sp., respectively. Pythium deliense was mostly effected by FP57 (59.80%), FP52 (57.80%) and FP56 (57.60%). While isolates FP35 and FP57 provided protection over 70% against all three pathogens in a climate chamber, they were as effective as commercial fungicides (Vitavax and Maxim) and biofungicide (Subtilex) and shown promising results.

Monitoring Lysobacter capsici AZ78 using strain specific qPCR reveals the importance of the formulation for its survival in vineyards.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application.

Antagonistic yeasts and thermotherapy as seed treatments to control Fusarium fujikuroi on rice

Bakanae disease, caused by Fusarium fujikuroi, is the most important seedborne disease of rice. Biological control and physical treatments can be effective tools to control seedborne diseases. Sixty-two isolates of yeasts and yeast-like fungi were obtained from different rice seeds. Four yeast isolates were selected in dual culture assays for mycelial growth inhibition, and in seed tests for reduction of infection rate. The isolates R23 and R26 were identified as Metschnikowia pulcherrima, the isolate R9 as Pichia guilliermondii, and the isolate SB1 as Sporidiobolus pararoseus. Rice seeds treated with P. guilliermondii R9, M. pulcherrima R23 and R26 significantly reduced the infection rate of F. fujikuroi, compared to some commercial biofungicides. The four selected yeasts reduced the bakanae disease severity in rice plants grown in greenhouse trials. Antagonist seed dressing resulted in reduction of the disease index from 93.0% in the untreated control to 20.0% in P. guilliermondii R9 treated seeds, and to 28.5% in M. pulcherrima R23 treated seeds. Selected antagonists were also used in combination with thermotherapy, which contributed to increase their efficacy. Thus, P. guilliermondii R9 and M. pulcherrima R23 combined with thermotherapy at 60°C for 10min decreased the bakanae disease index below 5%, and improved the seed germination rate compared to the single treatments, showing a seed priming effect. This is the first report about the use of antagonistic yeasts for seed dressing of rice to control F. fujikuroi on rice seeds, and biological treatment may be improved through combination with thermotherapy.

Influence of endophytic strains of the bacterium Bacillus subtilis on cell number in monocultures of green algae

Effect of several endophytic bacterial strains of Bacillus subtilis on growth of Scenedesmus quadricauda and Chlorella vulgaris algae was investigated. Introduction of bacillus strains into the algal cultures diminished the cell number of microalgae. The cells of C. vulgaris exhibited equal sensitivity to all the endophyte strains examined. The number of S. quadricaudas cells reduced to larger extent by the 7th day of algal culturing in the presence of B. subtilis 11V or 11VM strains than in the presence of B. subtilis 26D strain, the latter strain being the main component of the commercial biofungicide Fitosporin-M. Washed bacterial cells inactivated by autoclaving did not reduce the number of C. vulgaris cells. The bacterial cells that were autoclaved without separation from the culture liquid, as well as the autoclaved supernatant of cell suspension exerted the same inhibitory effect as living bacterial cells. The decrease in the algal cell number under the action of endophytic bacillus strains is presumably due to thermostable biologically active substances synthesized and secreted by the bacillus strains.