Dual Culture Method Research Articles

Metabolomic profiling and its association with the bio-efficacy of Aspergillus niger strain against Fusarium wilt of guava.

Bio-control agents are the best alternative to chemicals for the successful management of plant diseases. The fungus Aspergillus niger is known to produce diverse metabolites with antifungal activity, attracting researchers to exploit it as a bio-control agent for plant disease control. In the present study, 11 A. niger strains were isolated and screened for their antagonism against the guava wilt pathogen under in vitro and in planta conditions. Strains were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS), β-tubulin, and calmodulin genes. The strains were evaluated through dual culture, volatile, and non-volatile methods under an in vitro study. AN-11, AN-6, and AN-2 inhibited the test pathogen Fusarium oxysporum f. sp. psidii (FOP) at 67.16%, 64.01%, and 60.48%, respectively. An in planta study was conducted under greenhouse conditions with 6 months old air-layered guava plants (var. Allahabad Safeda) by pre- and post-inoculation of FOP. The AN-11 strain was found to be effective under both pre- and post-inoculation trials. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis was carried out to characterize the volatile compounds of the most potential strain, A. niger. The hexane soluble fraction showed the appearance of characteristic peaks of hexadecenoic acid methyl ester (4.41%), 10-octadecanoic acid methyl ester (3.79%), dodecane (3.21%), undecane (3.19%), gibepyrone A (0.15%), 3-methylundecane (0.36%), and citroflex A (0.38%). The ethyl acetate fraction of the bio-control fungi revealed the occurrence of major antifungal compounds, such as acetic acid ethyl ester (17.32%), benzopyron-4-ol (12.17%), 1,2,6-hexanetriol (7.16%), 2-propenoic acid ethanediyl ester (2.95%), 1-(3-ethyloxiranyl)-ethenone (0.98%), 6-acetyl-8-methoxy dimethyl chromene (0.96%), 4-hexyl-2,5-dihydro dioxo furan acetic acid (0.19%), and octadecanoic acid (1.11%). Furthermore, bio-control abilities could be due to hyper-parasitism, the production of secondary metabolites, and competition for sites and nutrients. Indeed, the results will enrich the existing knowledge of metabolomic information and support perspectives on the bio-control mechanism of A. niger.

Antifungal Activity of Bioactive Compounds Produced by the Endophytic Fungus Paecilomyces sp. (JN227071.1) against Rhizoctonia solani.

Biologically active natural compounds are molecules produced by plants or plant-related microbes, such as endophytes. Many of these metabolites have a wide range of antimicrobial activities and other pharmaceutical properties. This study aimed to evaluate (in vitro) the antifungal activities of the secondary metabolites obtained from Paecilomyces sp. against the pathogenic fungus Rhizoctonia solani. The endophytic fungus Paecilomyces was isolated from Moringa oleifera leaves and cultured on potato dextrose broth for the production of the fungal metabolites. The activity of Paecilomyces filtrate against the radial growth of Rhizoctonia solani was tested by mixing the filtrate with potato dextrose agar medium at concentrations of 15%, 30%, 45%, and 60%, for which the percentages of inhibition of the radial growth were 37.5, 50, 52.5, and 56.25%, respectively. The dual culture method was conducted on PDA medium to observe the antagonistic nature of the antibiotic impacts of Paecilomyces sp. towards the pathogenic fungus. The strength of the antagonistic impacts was manifested by a 76.25% inhibition rate, on a scale of 4 antagonistic levels. Ethyl acetate extract of Paecilomyces sp. was obtained by liquid-liquid partition of the broth containing the fungus. Gas chromatography-mass spectrometry (GC-MS) analysis identified the presence of important chemical components e.g., (E) 9, cis-13-Octadecenoic acid, methyl ester (48.607), 1-Heptacosanol, 1-Nonadecene, Cyclotetracosane (5.979), 1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester, di-sec-butyl phthalate (3.829), 1-Nonadecene, n-Nonadecanol-1, Behenic alcohol (3.298), n-Heptadecanol-1, 1-hexadecanol, n-Pentadecanol (2.962), Dodecanoic acid (2.849), 2,3-Dihydroxypropyl ester, oleic acid, 9-Octadecenal, and (Z)-(2.730). These results suggest that secondary metabolites of the endophytic Paecilomyces possess antifungal properties and could potentially be utilized in various applications, such as environmental protection and medicine.

Molecular diagnosis of red rot of sugarcane in Pakistan and in vitro control of isolated pathogen using rhizobacteria

Sugarcane (Saccharum officinarum L.) is a key cash crop, and its production is influenced by a variety of phytopathogens in different parts of the world. During consecutive field surveys, sugarcane stalks with red rot symptoms were observed in three provinces of Pakistan (Punjab, Sindh, and Khyber Pakhtunkhwa). Cane samples with visible symptoms were plated on Potato Dextrose Agar (PDA) media. Morphological and microscopic observations identified this pathogen as Colletotrichum falcatum. Comparative molecular study of amplified sequences of β-tubulin (Bt) and Actin (ACT) genes showed 100% similarity and ITS sequence showed 99% similarity with C. falcatum. For in vitro control of red rot, eight bacterial strains from the rhizosphere of sugarcane were isolated. These strains showed variable growth inhibition of C. falcatum in dual culture method. Among all tested strains, Bacillus amyloliquefaciens and B. altitudinis exhibited best antifungal activities. Based on these results, tested bacterial strains (B. amyloliquefaciens and B. altitudinis) can be recommended as effective biocontrol agents to manage red rot disease of sugarcane.

Biological Control of Brassicales Ring Spot Disease Caused by Alternaria brassicicola Using Antagonistic Yeasts

T his research investigated the biological control of Ring Spot Disease of Brassicales caused by Alternaria brassicicola, which is an agriculturally important pathogen, using antagonistic yeasts. One hundred fifteen yeasts were isolated from 38 fruits and vegetable leaves. A total of twenty-nine yeast isolates showed the inhibitory activity of A. brassicicola more than a value of 50% by the dual culture method. The result showed that three antagonistic yeast isolates Y107, Y123, and Y16 showed high inhibitory percentages at 75.00%, 73.52%, and 70.33%, respectively. Interestingly, these yeasts inhibited spore germination of A. brassicicola after 12 h within the range of 80.90 to 90.26%. Subsequently, the efficacy of the selected antagonistic yeast against Ring Spots Disease on cabbage seedlings (Brassica oleracea L. var. capitata) and Pak choi (Brassica chinensis L. var. chinensis) was tested in the greenhouse. The result indicated that antagonistic yeast isolate Y107 inhibited the disease at 72.75% and 71.42% in Cabbage seedlings and Pak choi, respectively. However, the efficacy of isolates Y123 and Y16 were not significant in disease inhibition in both Cabbage seedlings (66.67% and 66.08%) and Pak choi (69.83% and 68.92%). Additionally, the antagonistic yeast isolates were identified by phylogenetic analysis of the D1/D2 domain of the large subunit ribosomal RNA (LSU) gene. Yeast isolates Y16 and Y123 were identified to Kurtzmaniella quercitrusa, while isolate Y107 was Hanseniaspora thailandica.

In Vitro Efficacy Of Fungicides And Bioagents Against Colletotrichum Capsici Causing Leaf Spot Of Turmeric

Colletotrichum leaf spot is always regarded as a limiting factor for quantitative and qualitative losses of turmeric throughout the country. As a result, the current study on the efficacy of various fungicides and bio-agents against C. capsici under laboratory conditions was conducted to determine the most effective treatments. Using the poison food method, six different fungicides were tested in vitro against C. capsici. Among six fungicides tested, Tebuconazole 5.4 w/w FS, Carbendazim 12% + Mancozeb 63% WP, Azoxystrobin 18.2 w/w + Difenoconazole 11.4 w/w SC, Carboxin 37.5% + Thiram 37.5% DS inhibited 100% mycelial growth inhibition of C. capsici. The bio-agent was tested against C. capsici using the dual culture method. Trichoderma harzianum was found to be the most effective as a bioagent, with a maximum inhibition of mycelial growth of (68.66%) followed by T. asperellum with (68.36%), Pseudomonas fluroscens (64.29%) and Bacillus subtilis (60.24%).

KOMPATIBILITAS BEBERAPA SPESIES Bacillus SEBAGAI BIOAKTIVATOR PUPUK ORGANIK HAYATI

Bacteria is a potential agent in biological organic fertilizer”s process. The rate of decomposition of organic matter in biological organic fertilizers process depends on the role of funtional bacteria, the type of organic matter and the microenvironment. Bacteria that are inoculated into biological organic fertilizer’s matter must be synergistic among them, so that their role as as bioactivators more efficient. A compatibility experiment among the bacteria isolates used as bioactivators have to be done in order to obtain an effective and efficient biological organic fertilizer. The objevctive of the research is to obtain information on the compatible nature of the bacterial isolates. The research was conducted at the Plant Protection Laboratory of the Payakumbuh State Agricultural Polytechnic, West Sumatra from July to August 2022. The research method began with the rejuvenation bacterial isolates of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis on NA media, then continued with a compatibility experiment using the dual culture method on NA and King’s B medium, so there were a total of 12 treatments (single and combination) with three replications. The results of the compatibility experiment on the three Bacillus bacteria did not form a halo inhibition zone and were compatible with the compatibility index value (IK) 0.67-1 on NA medium and 0.90-1.00 on King’s B medium, meaning that the growth between the three Bacillus bacteria did not inhibit each other, so that all Bacillus bacteria could be used as a bioactivator simultaneously in biological organic fertilizer’s process.

Soil Fungistasis against Fusarium Graminearum under Different tillage Systems

The establishment of the harmful pathogen Fusarium graminearum in different agroecosystems may strongly depend on the ability of the soils to suppress its development and survival. This study aimed to evaluate the influence of different soil tillage systems (i.e., conventional tillage, reduced tillage and no-tillage) on soil fungistasis against F. graminearum. Soil samples were collected three times during the plant growing season in 2016 and 2017 from a long-term, 20-year soil tillage experiment. The F. graminearum in the soil samples was quantified by real-time qPCR. The soil fungistasis was evaluated by the reduction in the radial growth of F. graminearum in an in vitro assay. The antagonistic activity of the soil bacteria was tested using the dual culture method. The F. graminearum DNA contents in the soils were negatively correlated with soil fungistasis (r = -0.649 *). F. graminearum growth on the unfumigated soil was reduced by 70-87% compared to the chloroform fumigated soil. After the plant vegetation renewal, the soil fungistasis intensity was higher in the conventionally tilled fields than in the no-tillage. However, no significant differences were obtained among the tillage treatments at the mid-plant growth stage and after harvesting. 23 out of 104 bacteria isolated from the soil had a moderate effect, and only 1 had a strong inhibitory effect on the growth of F. graminearum. This bacterium was assigned 100% similarity to the Bacillus amyloliquefaciens Hy7 strain (gene bank no: JN382250) according to the sequence of the 16S ribosome subunit coding gene. The results of our study suggest that the presence of F. graminearum in soil is suppressed by soil fungistasis; however, the role of tillage is influenced by other factors, such as soil biological activity, type and quantity of plant residues and environmental conditions.

Inhibition of Lasiodiplodia pseudotheobromae Causing Fruit Rot Disease of Longan by Using Antagonistic Bacillus siamensis RFCD306

P ostharvest fruit rot disease of longan (Dimocarpus longan Lour.) caused by Lasiodiplodia pseudotheobromae is one of the most devastating problems for longan production in Thailand. To reduce the use of chemicals, the endophytic bacterium was isolated from healthy longan fruit for using as a biological control agent. The antagonistic bacterium Bacillus siamensis isolate RFCD306 which was identified based on morphological, biochemical and molecular characterization. It showed the antifungal effect with 94.40% of fungal growth inhibition which was conducted by a dual culture method. The efficacy of B. siamensis RFCD306 on the inhibition of fungal growth and conidial germination was observed under the scanning electron microscope (SEM) and revealed the abnormal germination of conidial and hyphal growth. Moreover, four antibiotic synthesis-related genes consisting of srfA, ituA, bacD and bymA were detected from B. siamensis RFCD306 by a polymerase chain reaction (PCR) technique. These genes might be involved in the antifungal activity of the antagonistic bacterium. Furthermore, the disease control efficiency of B. siamensis RFCD306 was evaluated on harvested longan fruits and showed that the bacterium decreased the incidence of fruit rot disease. Therefore, this study represented the effective B. siamensis RFCD306 for using as a biological agent in controlling postharvest fruit rot disease of longan.

Cultural Characterization and Antagonistic Activity of Cladobotryum virescens against Some Phytopathogenic Fungi and Oomycetes

In this study, the characteristic growth of Cladobotryum virescens on nine culture media was analyzed. The growing behavior of this fungus was dependent on the culture medium. In vitro analysis showed that oat agar was better than other media tested with the highest conidia production. The antifungal activity against Fusarium chlamydosporum and Alternaria brassicicola was evaluated by the Dual Culture method. C. virescens displayed high activity against both pathogens acting through antibiosis and mycoparasitism. This effect was increased by a higher competitiveness of the strain for the substrate. Furthermore, the crude ethyl acetate extract of the culture broth was tested in vitro against Botrytis cinerea and Septoria tritici, as well as the hemibiotrophic oomycete Phytophthora infestans using a microtiter plate assay at different concentrations. The extract showed excellent inhibition even below 5 ppm. According to these results, we concluded that C. virescens can be considered as a potential biological control agent in agriculture. To the best of our knowledge, this is the first study to investigate C. virescens as a biocontrol agent for different diseases caused by five relevant pathogens that affect cereals and vegetables.

Trichoderma pubescens Elicit Induced Systemic Resistance in Tomato Challenged by Rhizoctonia solani.

Rhizoctonia solani causes severe diseases in many plant species, particularly root rot in tomato plants. For the first time, Trichoderma pubescens effectively controls R. solani in vitro and in vivo. R. solani strain R11 was identified using the ITS region (OP456527); meanwhile, T. pubescens strain Tp21 was characterized by the ITS region (OP456528) and two genes (tef-1 and rpb2). The antagonistic dual culture method revealed that T. pubescens had a high activity of 76.93% in vitro. A substantial increase in root length, plant height, shoot fresh and dry, and root fresh and dry weight was indicated after applying T. pubescens to tomato plants in vivo. Additionally, it significantly increased the chlorophyll content and total phenolic compounds. The treatment with T. pubescens exhibited a low disease index (DI, 16.00%) without significant differences with Uniform® fungicide at a concentration of 1 ppm (14.67%), while the R. solani-infected plants showed a DI of 78.67%. At 15 days after inoculation, promising increases in the relative expression levels of three defense-related genes (PAL, CHS, and HQT) were observed in all T. pubescens treated plants compared with the non-treated plants. Plants treated with T. pubescens alone showed the highest expression value, with relative transcriptional levels of PAL, CHS, and HQT that were 2.72-, 4.44-, and 3.72-fold higher in comparison with control plants, respectively. The two treatments of T. pubescens exhibited increasing antioxidant enzyme production (POX, SOD, PPO, and CAT), while high MDA and H2O2 levels were observed in the infected plants. The HPLC results of the leaf extract showed a fluctuation in polyphenolic compound content. T. pubescens application alone or for treating plant pathogen infection showed elevated phenolic acids such as chlorogenic and coumaric acids. Therefore, the ability of T. pubescens to inhibit the growth of R. solani, enhance the development of tomato plants, and induce systemic resistance supports the application of T. pubescens as a potential bioagent for managing root rot disease and productivity increase of crops.

Screening of antagonistic yeast strains for postharvest control of Penicillium expansum causing blue mold decay in table grape

Blue mold decay caused by Penicillium expansum is one of the most important postharvest diseases of grapes, leading to considerable economic losses. Regarding the increasing demand for pesticide-free foods, this study aimed to find potential yeast strains for biological control of blue mold on table grapes. A total of 50 yeast strains were screened for antagonistic activity against P. expansum using the dual culture method and six strains significantly inhibited the fungal growth. All six yeast strains (Coniochaeta euphorbiae, Auerobasidium mangrovei, Tranzscheliella sp., Geotrichum candidum, Basidioascus persicus, and Cryptococcus podzolicus) reduced the fungal growth (29.6–85.0%) and the decay degree of wounded grape berries inoculated with P. expansum while G. candidum was found to be the most efficient biocontrol agent. On the basis of antagonistic activity, the strains were further characterized by in vitro assays involving inhibition of conidial germination, production of volatile compounds, iron competition, production of hydrolytic enzymes, biofilm-forming capacity, and exhibited three or more putative mechanisms. To our knowledge, the yeasts are reported for the first time as potential biocontrol agents against the blue mold of grapes but more study is required to evaluate their efficiency related to field application.

Capability of Three Yeast Species in Suppressing Green Mold (Penicillium Digitatum) on Siam Citrus Fruit (Citrus Nobilis)

Green mold is one of the main diseases in citrus fruits caused by Penicillium digitatum . One of the managements that can be used to control this disease is by using yeasts as antagonistic agents. The objective of this study was to evaluate the ability of yeast to suppress Green mold caused by P. digitatum in siam citrus ( Citrus nobilis Lour.). The study was conducted using a completely randomized design. The treatments were the application of yeast, consisting of Aureobasidium pullulans isolate Dmg 11 DEP, Rhodotorula minuta isolate Dmg 16 BE, Candida tropicalis isolate Lm 13 BE, fungicide benomyl, and control. The results showed that A. pullulans isolate Dmg 11 DEP, C. tropicalis isolate Lm 13 BE, and R. minuta isolate Dmg 16 BE were able to suppress the growth of P. digitatum in vitro on dual culture method, with suppression levels ranged from 14.64%-21.02%. The greatest suppression was caused by R. minuta isolate Dmg 16 BE. The formation of volatile compounds test results were ranged from 14.51%-34%, and the highest suppression was on C. tropicalis isolate Lm 13 BE. Application of the yeasts on siam citrus fruit was able to reduce the pathogen’s growth, and the suppressions were 28.87% to 68.72%. R. minuta isolate Dmg 16 BE caused the highest suppression.

The inhibition of Fusarium wilt in Chili by Endophytic Fungi isolated from Green Betel (Piper betle L.) Leaf

Green Betel (Piper betle L.) leaves are often used as medicine by local people because they contain antibacterial and antifungal substances. These substances can be produced by plant metabolites and are also the results of metabolites produced by endophytic fungi. Endophytic fungus is an organism that is associated with healthy host tissues without causing disease symptoms. Endophytic fungi from betel leaf are used as biocontrol agents against Fusarium oxysporum causes wilt disease that attacks Chili (Capsicum annuum L.), which is one of the main agricultural commodities in Indonesia. Fusarium wilt disease is a disease that significantly reduces crop yields based on crop yields. This research was conducted in a chili farm in Cangkringan, Sleman, Yogyakarta. This study aimed to test the antagonism of endophytic fungi with the dual culture technique. Three types of green betel leaf endophytic fungi were used for dual culture test against Fusarium oxysporum. The observation result showed the highest inhibition ability of 73.37% dual culture method. The inhibitory mechanisms of green betel leaf endophytic fungal isolates include parasitism, antibiosis, and competition. Endophytic fungi of green betel leaf are effective as biocontrol agents of wilt Fusarium chili disease. ©2022 JNSMR UIN Walisongo. All rights reserved.

Screening of endophytic bacteria from potato tubers and their antagonistic activity against soil-borne potato pathogens

In order to appraise the bacterial endophyte communities that help resist disease in potato tuber, the separation, the population density, biodiversity and the antagonistic activity of endophytic bacteria, from the tuber peel of potato cultivars (Fontan90, Agria, Sante’a and Jeli89), were examined in the Fars province in Iran. In this study, the bacterial endophyte Colony Forming Units (CFU) were counted based on the most suitable dilution in petri dishes and expressed per g of wet weight of tuber tissue. The presence of bacteria was found mostly in the outer layer. A wide variety of endophyte species biodiversity was in Agria cultivar. To estimate the antagonistic effect of potato associated endophytic bacteria, 115 bacterial isolates were evaluated by dual culture method against main soil-borne potato pathogens Fusarium oxysporum, Rhizoctonia solani, Verticillium dahliae, Streptomyces scabies and Ralstonia solanacearum. Endophyte strains were identified based on physiological, morphological and chemical characteristics and the 16S rRNA gene sequence analysis. The highest degree of the inhibitory activity in all layers of potato cultivars was related to Bacillus subtilis, Bacillus mojavensis and Klebsiella variicola. Antagonistic activity of endophytic bacteria against the pathogens was significantly higher (p<0.01) in the examined strains from the outermost layer of tuber peel and decreased progressively toward the center of the tuber. In this research, Klebsiella variicola was reported as endophyte bacteria in the four commercial potato cultivars mentioned above, for the first time.

Characterization of the Mechanism of Action of Serratia rubidaea Mar61-01 against Botrytis cinerea in Strawberries.

Several bacterial strains belonging to Serratia spp. possess biocontrol capability, both against phytopathogens and human pathogenic species, thanks to the production of secondary metabolites, including as a red-pink, non-diffusible pigment, 2-methyl-3-pentyl-6-methoxyprodiginine (prodigiosin). Botrytis cinerea is the causal agent of gray mold, which is an economically relevant disease of many crops worldwide. Gray mold is normally controlled by chemical fungicides, but the environmental and health concerns about the overuse of pesticides call for environmentally friendly approaches, such as the use of biocontrol agents. In this study, the efficacy of a specific strain of Serratia rubidaea (Mar61-01) and its metabolite prodigiosin were assessed against B. cinerea under in vitro and in vivo conditions. This strain was effective against B. cinerea, and the effect of prodigiosin was confirmed under in vitro and in vivo conditions. The strain suppressed mycelial growth of B. cinerea (71.72%) in the dual-culture method. The volatile compounds produced by the strain inhibited mycelial growth and conidia germination of B. cinerea by 65.01% and 71.63%, respectively. Efficacy of prodigiosin produced by S. rubidaea Mar61-01 on mycelial biomass of B. cinerea was 94.15% at the highest concentration tested (420 µg/mL). The effect of prodigiosin on plant enzymes associated with induction of resistance was also studied, indicating that the activity of polyphenol oxidase (PPO), superoxide dismutase (SOD) and phenylalanine ammonia lyase (PAL) were increased when prodigiosin was added to the B. cinerea inoculum on strawberry fruits, while catalase (CAT) and peroxidase (POD) did not change. In addition, the volatile organic compounds (VOCs) produced by S. rubidaea Mar61-01 reduced mycelial growth and inhibited conidial germination of B. cinerea in vitro. The findings confirmed the relevant role of prodigiosin produced by S. rubidaea Mar61-01 in the biocontrol of B. cinerea of strawberries, but also indicate that there are multiple mechanisms of action, where the VOCs produced by the bacterium and the plant-defense reaction may contribute to the control of the phytopathogen. Serratia rubidaea Mar61-01 could be a suitable strain, both to enlarge our knowledge about the potential of Serratia as a biocontrol agent of B. cinerea and to develop new biofungicides to protect strawberries in post-harvest biocontrol.

Efficacy of Trichoderma isolates against Sclerotium rolfsii causing collar rot of lentil

Lentil collar rot disease caused by Sclerotium rolfsii Sacc. is an important disease causing significant yield loss in Nepal. Seven Trichoderma spp. isolates collected from different regions of Nepal were tested for their capacity to inhibit mycelial growth of Sclerotium rolfsii in in-vitro condition. Experiment was conducted in Completely Randomized Design with four replications in the plant pathology laboratory of Grain Legumes Research Program, Khajura, Banke during 2020. Dual culture method was performed to assess the efficacy of Trichoderma isolates. Trichoderma spp. isolates inhibited mycelial growth of S. rolfsii to various degrees ranging from 48.33-72.47% after 120 hours of inoculation. Maximum colony inhibition of S. rolfsii (72.47%) was obtained by Kapilvastu isolate followed by Nepalgunj isolate (67.72%). Minimum colony inhibition of S. rolfsii (48.33%) was obtained by Mangalpur, Chitwan isolate followed by Rampur, Chitwan isolate (49.33%). Among seven Trichoderma isolates, two isolates (Kapilbastu and Nepalgunj) showed good antagonistic activity against S. rolfsii and were also highly competitive in in-vitro condition. These isolates would therefore be useful in biological disease management.

Molecular characterization of indigenous microbes and its potential as a biological control agent of Fusarium stem rot disease (Fusarium verticillioides) on maize

Fusarium stem rot disease caused by Fusarium verticillioides has become one of the most serious issues confronting Indonesian farmers in recent years. An alternative option for suppressing this disease is to use indigenous microbes as an eco-friendly method to reduce synthetic fungicides. The objective of the research was to identify the molecular characteristics and effectiveness of an indigenous microbial consortium in controlling Fusarium stem rot disease. Identification of indigenous microbes is carried out based on molecular characters using universal primers, namely ITS-1/ITS-4 for fungi and 27F/1492R for bacteria. Nucleotide sequences were analyzed using Bioedit 7.2 version and MEGAX software. In vitro testing was carried out using the dual culture method for indigenous fungi and the disc diffusion method for indigenous bacteria. Meanwhile, in planta testing was conducted by evaluating a consortium of fungi and bacteria to control F. verticillioides in the field using a randomized block design with three replications, followed by a 5% DMRT test. The use of universal primer pairs ITS-1/ITS-4 and 27F/1492R succeeded in amplifying DNA bands of indigenous microbial isolates measuring ±600 bp and ±1465 bp, respectively. S6 and S9 bacterial isolates were identified as Bacillus cereus. JRP 7 and SEDF 6A isolates were identified as Trichoderma asperellum and JRP 10 isolate was identified as Penicillium raperi. All identification resulted in homology of >99%. The in vitro inhibitory reactions of isolates JRP 7, JRP 10, SEDF 6A, S6, and S9 against F. verticillioides were >60%. Disease severity of B6B9C10, B6B9C6, B6B9C7, B9C6C7, B9C7C10, and C6C7C10 treatments significantly proved their ability to control F. verticillioides in the field with a lower percentage of disease severity than positive controls, which are 23.33%, 18.89%, 23.33%., 21.85%, 14.07%, and 15.93%, respectively. The B9C7C10 consortium (S9 + JRP 7 + JRP 10 isolates) containing three species of indigenous microbes, i.e. B. cereus, T. asperellum, and P. raperi is the most effective at controlling F. verticillioides and may be developed for use as biopesticide products.

Micromycetes Trichoderma for plants protection against alternariosis

Currently there is an increased presence of Alternaria sp. mycotoxins in raw food materials and foods. In order to reduce the development of Alternaria fungi and to reduce their harmfulness, it is important to use biological preparations on the base of the microorganisms for plant protection. The aim of the research is to select strains of Trichoderma sp. fungi promising to control alternariosis in plants crops. The antagonistic activity of the mycelial culture of Trichoderma towards Alternaria was studied by dual culture method. The antifungal activity of the culture liquid was evaluated by agar diffusion method. The culture liquids containing metabolites of three strains, inhibited the growth of the pathogen by forming inhibition zones with a radius of 13-16 mm. Germination of wheat seeds on infectious background of soil contamination with the pathogen A.alternata identified micromycetes to create a biological product against alternariosis. Biological effectiveness of isolates 2N and 13N used for seeds protection against Alternaria is 87.3% and 83.6%, respectively.

Screening of antagonistic strains <i>Trichoderma</i> Pers. towards causative agent of root rot in apples of genus Fusarium Link.

In recent years, in the south of Russia, fungi of the genus Fusarium Link (1809) constitute a special danger to such important agricultural perennial crops as the apple tree. Тhese fungi cause root rot, a harmful disease, which weakens the vascular organs, leading to a significant decrease in yield and even death of the plant. A literature review showed that the genus Trichoderma has great potential for the integrated protection of apple trees. In this regard, due to the lack of registered microbiological agents for the control of fusarium root rot in apple trees, the search for antagonistic strains for biotechnological control of this disease becomes relevant. In this work, the parasitic activity of strains of the genus Trichoderma against the causative agents of root rot in apple trees, namely Fusarium sporotrichioides and Fusarium oxysporum, was studied in vitro. Тhe research was carried out in 2020–2021 in the Laboratory of biotechnological control of phytopathogens and phytophages of the FGBSO “North Caucasian Federal Scientific Center of Horticulture, Viticulture and Winemaking”. Тhe research was focused on monoconidial strains of the causative agent of apple root rot: F. sporotrichioides (strain RR20XXIV/4.3) and F. oxysporum (strain RR20XXV/6.2), as well as 7 strains of the genus Trichoderma having expected antagonistic activity. Тhe activity of strains of the genus Trichoderma against fungi of the genus Fusarium was determined by the dual culture method using joint splicing on the PGA medium (potato-glucose agar). Тhe Trichocin, SP (titer 1010 CFU/g Trichoderma harzianum, strain G-30 VISR), registered against root rot for a number of crops, was used as a standard. A monoculture of the test object (strains of the genus Fusarium) was used as a control. Out of the 7 strains of antagonistic fungi, strains 20I/1 and 20II/2 showed the greatest activity against F. sporotrichioides by a dual mechanism of action, which involves competition for a nutrient medium and fungicidal parasitic antagonism; the latter strain showed hyperparasitism. Antibiotic (strain 20XIV/5) and alimentary antagonisms (strain 20II/3.2) were also detected. Strain 20XIV/5 showed antibiotic antagonism against F. oxysporum, having higher activity than the standard; strain 20I/1 showed alimentary antagonism. Тhe remaining antagonist strains showed competitive interactions for the environment.

Selection and characterization of a yeast strain for the suppression of brown spot on Tru Long pummelo (Citrus maxima)

Tru Long pummelo is an endemic plant of Quang Nam province with high economic value. However, many different diseases, especially brown spot disease caused by Alternaria fungus on pummelos, have affected the quality of pummelos. Pesticides are overused and misused for the environment and human health. Therefore, there is an urgent need to reduce the use of synthetic chemicals. Biological control offers an alternative to the use of pesticides. Eight yeast strains have been isolated from healthy leaves, pummelo fruits, and healthy mulberries. They were screened for biological activity against Alternaria sp. by the dual culture method. The inhibitory potency ranged from 10.46% to 59.86%. The strain with the highest percentage (59.86%) was identified as Candida tropicalis using sequence analysis of the ITS region.