Biological control of tomato early blight in Pakistan using local rhizobacteria.

BACKGROUND: The biocontrol potential of soil microbes can reduce the extensive use of hazardous synthetic fungicides. This study was designed to find a strain of rhizobacteria indigenous to Pakistan with potential biocontrol against early blight of tomato caused by Alternaria solani and to characterize its biocontrol mechanisms. RESULTS: Among 88 strains tested for antagonism against A. solani on agar media, S27, Dt10 and 423, identified by 16S rRNA sequencing as strains of Bacillus amyloliquefaciens, B. cereus and Stenotrophomonas rhizophila, respectively, were the most inhibitory. When applied to detached tomato leaflets in Petri dish assays, the strains reduced lesion development by over 30% compared to the control. In greenhouse pot trials, the bacterial strains reduced early blight severity by over 50%. In three field trials, all three strains applied to tomato foliage slowed early blight disease progress and reduced disease severity, with B. amyloliquefaciens S27 reducing the area under the disease progress curve by up to 70%. All three strains showed protease, catalase and oxidase activities in vitro, but none produced ß-1,3-glucanase and only B. cereus Dt10 showed slight chitinase activity. In a greenhouse experiment in which the bacteria were applied to tomato foliage prior to pathogen inoculation, bacteria-treated leaves had higher ß-1,3-glucanase and chitinase levels than leaves inoculated only with the pathogen, indicating priming induction of response. CONCLUSION: Three rhizobacteria strains have the potential to control early blight of tomato under Pakistan's growing conditions, with B. amyloliquefaciens S27 being the most promising candidate for commercial development. Antagonism and induction of the priming response may be mechanisms of biocontrol by the bacterial strains. © 2023 Society of Chemical Industry.

Synthesis of Polyaniline Coated Magnesium and Cobalt Oxide Nanoparticles through Eco-Friendly Approach and Their Application as Antifungal Agents.

Plant-mediated synthesis of nanoparticles exhibits great potential to minimize the generation of chemical waste through the utilization of non-toxic precursors. In this research work, we report the synthesis of magnesium oxide (MgO) and cobalt oxide (Co3O4) nanoparticles through a green approach using Manilkara zapota leaves extract, their surface modification by polyaniline (PANI), and antifungal properties against Aspergillus niger. Textural and structural characterization of modified and unmodified metal oxide nanoparticles were evaluated using FT-IR, SEM, and XRD. The optimal conditions for inhibition of Aspergillus niger were achieved by varying nanoparticles' concentration and time exposure. Results demonstrate that PANI/MgO nanoparticles were superior in function relative to PANI/Co3O4 nanoparticles to control the growth rate of Aspergillus niger at optimal conditions (time exposure of 72 h and nanoparticles concentration of 24 mM). A percentage decrease of 73.2% and 65.1% in fungal growth was observed using PANI/MgO and PANI/Co3O4 nanoparticles, respectively, which was higher than the unmodified metal oxide nanoparticles (67.5% and 63.2%).

First report of Alternaria alternata causing Alternaria leaf spot of Fig in Pakistan.

Fig (Ficus carica) is a species of flowering plants within the mulberry family. During June 2020, leaf spots were observed on several fig plants (31°26'15.0"N 73°04'25.6"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, oval to circular, light brown, sunken spots that were uniformly distributed on the leaves. Spots gradually enlarged and coalesced into circular to irregular dark brown to black spots that could be up to 3cm diam. with no or small sized fruit. Disease incidence was approximately 25%. To identify the causal agent of the disease, 15 symptomatic leaves were collected. Small pieces from all diseased samples were removed from the margin between healthy and diseased tissues were surface disinfested in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Fungal isolation on PDA medium frequency was 95% from diseases leaves. Morphological observations were made on 7- day- old single-spore cultures. The colonies initially appeared light grayish which turned sooty black in color. All fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 9 to 25 µm × 5 to 10 µm (n = 40) with longitudinal and transverse septa. The morphological characters matched those of Alternaria alternata (Simmons et al. 2007). Genomic DNA of a representative isolate (FG01-FG03) was extracted using DNAzol reagent (Thermo Fisher Scientific MA, USA) and PCR amplification of the internal transcribed spacer (ITS) rDNA region, was performed with primers ITS1/ITS4 (White et al. 1990), partial RNA polymerase II largest subunit (RPB2) with RPB2-5F/RPB2-7cR (Liu et al. 1999) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene regions was performed with gpd1/gpd2 (Berbee et al. 1999). The obtained sequences were deposited in GenBank with accession numbers MW692903.1 to MW692905.1 for ITS-rDNA gene, MZ066731.1 to MZ066733.1 for RPB2 and MZ066728.1 to MZ066730.1 for GAPDH. BLASTn analysis showed 100% identity with the submitted sequences of A. alternata for ITS rDNA, RPB2, and GAPDH. To confirm pathogenicity, 2-month-old 15 healthy potted F. carica plants were sprayed at true leaf stage with conidial suspension by using an atomizer in a greenhouse. Each representative A. alternata isolate (FG01-FG03) was inoculated on every three plants with conidial suspensions (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) whereas, three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. All plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues and reisolation frequency was 100%. Re-isolated fungal cultures were again morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported cause fruit disease of fig in Pakistan and California, USA (Alam et al. 2021; Latinovic et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot on common fig in Pakistan. In Pakistan, fig is widely grown for drying, and this disease may represent a threat to fig cultivation.

In situ Impact of the Antagonistic Fungal Strain, Trichoderma gamsii T30 on the Plant Pathogenic Fungus, Rhizoctonia solani in Soil.

Rhizoctonia solani is a soil-borne fungus causing a wide range of plants diseases. Trichoderma gamsii strain T30 has previously been reported as antagonistic against R. solani. Although there are a few studies about the influence of Trichoderma strains on the R. solani density in a pathosystem in the presence of plant hosts, this report for the first time comprehensively describes in situ effects of a T. gamsii strain on the population density of R. solani in the soil microcosmic conditions. The population dynamics of R. solani were followed in the autoclaved and non-autoclaved soils in artificially prepared microcosms up to day 25 after co-inoculation with T. gamsii in the variable ratios (R1/T1; R1/T0.1; R1/T0.01 of R. solani/T. gamsii). The population density of R. solani was evaluated by qPCR. In the autoclaved soil, target DNA copies of R. solani increased in the control samples from 1 × 105 to 6.5 × 106. At R1/T0.01, the number of target DNA copies were not significantly changed until day 11; however, it decreased by around five times at day 25. At R1/T0.1 and R1/T1, the number of DNA copies was reduced to 2.1 × 106 and 7.6 × 105 at day 11, respectively and the reduction was as much as 17 times at day 25. In the non-autoclaved soil, the number of the fungal cells decreased at day 25 whether inoculated or not with Trichoderma indicating a general suppression by the soil microbiome. In brief, T. gamsii significantly inhibited the growth of R. solani in the soil in situ and there was a general suppressive effect of the natural microbiome.Rhizoctonia solani is a soil-borne fungus causing a wide range of plants diseases. Trichoderma gamsii strain T30 has previously been reported as antagonistic against R. solani. Although there are a few studies about the influence of Trichoderma strains on the R. solani density in a pathosystem in the presence of plant hosts, this report for the first time comprehensively describes in situ effects of a T. gamsii strain on the population density of R. solani in the soil microcosmic conditions. The population dynamics of R. solani were followed in the autoclaved and non-autoclaved soils in artificially prepared microcosms up to day 25 after co-inoculation with T. gamsii in the variable ratios (R1/T1; R1/T0.1; R1/T0.01 of R. solani/T. gamsii). The population density of R. solani was evaluated by qPCR. In the autoclaved soil, target DNA copies of R. solani increased in the control samples from 1 × 105 to 6.5 × 106. At R1/T0.01, the number of target DNA copies were not significantly changed until day 11; however, it decreased by around five times at day 25. At R1/T0.1 and R1/T1, the number of DNA copies was reduced to 2.1 × 106 and 7.6 × 105 at day 11, respectively and the reduction was as much as 17 times at day 25. In the non-autoclaved soil, the number of the fungal cells decreased at day 25 whether inoculated or not with Trichoderma indicating a general suppression by the soil microbiome. In brief, T. gamsii significantly inhibited the growth of R. solani in the soil in situ and there was a general suppressive effect of the natural microbiome.

Influence of Frequently Used Chemical Insecticides on Mycoflora Carried by Common Housefly, Musca domestica L.

The housefly, Musca domestica L. (Diptera: Muscidae), is a major medical and veterinary insect pest. It serves as a vector of many pathogenic microorganisms causing spoilage of food and diseases in human and animals. Use of chemical insecticides is adapted as a principal tool to manage housefly. Insecticides have many unforeseen ecological consequences including effects on non-target organisms. In the present study, we have assessed the effects of 10 different synthetic insecticides on the growth of mycoflora associated with the external body of the housefly by using poison food technique. Our results reveled that all synthetic insecticides enhanced the growth. Surprisingly, in most of the cases, mycelial growth of fungi was significantly increased at high concentration as compared with lower concentration. This study provides useful information about the dangerous effects of synthetic insecticides on environment by increasing the spread of various non-target pathogenic, mycotoxigenic, and food spoiling fungi, carried by houseflies.

Antifungal Potential of Indigenous Medicinal Plants against Myrothecium Leaf Spot of Bitter Gourd ( Momordica charantia L.)

ABSTRACT Bitter gourd is of great importance due to its usage against the treatment of numerous ailments in human beings. A comprehensive survey at four localities of Southern Punjab, Pakistan was carried out to determine the severity of Myrothecium leaf spot. Maximum disease severity was at C1 (Chak 11/NP) and least at C2 (Kot Mehtab). Among isolated species Myrothecium roridum was found more prevalent and pathogenic as compared to M. verrucaria. Antifungal activity using solvent extracts of five medicinal plants (Mangifera indica, Melia azedarach, Nicotiana tabacum, Moringa oleifera and Eucalyptus globosum) were evaluated against isolated species by agar well diffusion method at various concentrations (0.01, 0.10, 1.0 and 10.0 µg / mL). N. tabacum revealed maximum zone size (13.40 mm and 8.28 mm) with ethanol and chloroform solvents respectively followed by M. azedarach (9.00mm and 6.48mm). However, least inhibition was observed with ethanol and chloroform extracts of E. globosum (6.04mm and 3.88mm zone size respectively). Ethanol extracts showed highest activity when compared to chloroform extracts. Qualitative phytochemical analysis showed that all the selected plants are rich in chemical compounds such as alkaloids, terpenoids, flavonoids and phenols whereas Saponins was only present in N. tabacum while absent in rest of the extracts.