Evaluating the Antifungal Activity of Some Traditional Medicinal Plant Extracts against Alternaria solani (Tomato Early Blight Pathogen) in Ethiopia

Background: Tomato (Lycopersicon esculentum Mill.) belongs to the family Solanaceae. In Ethiopia, control of early blight is largely dependent on fungicidal application. There is a research need to identify effective botanical extracts to control Alternaria solani that cause early blight of tomato and for evaluation of plant extracts through different solvents on the target pathogen. Methods: In vitro experiment was conducted to evaluate the effectiveness of crude extracts of 16 selected medicinal plants against Alternaria solani. Thus, crude extracts were extracted from medicinal plants with different solvents (methanol, ethanol and petroleum at (25%, 50% and 100%) concentrations. The Alternaria solani was isolated from infected tomato leaves showing early blight symptoms. Evaluation of plant extracts was carried out against Alternaria solani using food poisoned technique on PDA. Result: Results showed that most of the methanolic extract plants were showed significant inhibition of the mycelial growth as compared to ethanolic and petroleum ether extracts. A higher rate of mycelial reduction was recorded by ethanol extracts of Allium sativum at all concentrations (100%) followed by methanol extracts of Allium sativum at 25%, 50%, 100% concentration (90.02%, 97.01%, 100% respectively). The effectiveness of extracts against Alternaria solani depends on use at the higher concentrations and various solvents. For crude extracts that have shown higher inhibitory effects against Alternaria solani in vitro conditions, actual chemical compounds should be identified. Furthermore, it is also important to evaluate these plants on other microbes, study to test in vivo and to assess their real potential field condition wherever early blight is an important disease of tomato.

Mechanism of a Volatile Organic Compound (6-Methyl-2-Heptanone) Emitted From Bacillus subtilis ZD01 Against Alternaria solani in Potato

The antagonistic mechanisms of soluble non-volatile bioactive compounds, such as proteins and lipopeptides emitted from Bacillus have been widely studied. However, there are limited studies on the antifungal mechanisms of volatile organic compounds (VOCs) produced by Bacillus against plant fungal diseases. In this study, the antagonistic mechanisms of one specific VOC, 6-methyl-2-heptanone, against Alternaria solani were investigated. To optimize the extraction conditions of headspace solid-phase microextraction, a 50/30-μm divinylbenzene/carboxen/polydimethylsiloxane fiber at 50°C for 40 min was used. For gas chromatography-mass spectrometry using a free fatty acid phase capillary column, 6-methyl-2-heptanone accounted for the highest content, at 22.27%, of the total VOCs from Bacillus subtilis ZD01, which inhibited A. solani mycelial growth strongly in vitro. Therefore, 6-methyl-2-heptanone was selected as the main active chemical to elucidate the action mechanisms against A. solani. Scanning and transmission electron microscopy analyses revealed that after exposure to an EC50 dose of 6-methyl-2-heptanone, A. solani hyphal cells had a wide range of abnormalities. 6-Methyl-2-heptanone also caused the capture of cellular fluorescent green label and the release of adenosine triphosphate (ATP) from outer membranes A. solani cells, which may enhance 6-methyl-2-heptanone ability to reach the cytoplasmic membrane. In addition, 6-methyl-2-heptanone showed strong inhibitory effect on A. solani conidial germination. It also damaged conidial internal structures, with the treated group having collapsed shrunken small vesicles as observed by transmission electron microscopy. Because 6-methyl-2-heptanone showed strong effects on mycelial integrity and conidial structure, the expression levels of related pathogenic genes in A. solani treated with 6-methyl-2-heptanone were investigated. The qRT-PCR results showed that transcriptional expression levels of slt2 and wetA genes were strongly down-regulated after exposure to 6-methyl-2-heptanone. Finally, because identifying the functions of pathogenic genes will be important for the biological control of A. solani, the wetA gene was identified as a conidia-associated gene that plays roles in regulating sporulation yield and conidial maturation. These findings provide further insights into the mechanisms of VOCs secreted by Bacillus against A. solani.

Genome-Wide Analysis of TIR-NBS-LRR Gene Family in Potato Identified StTNLC7G2 Inducing Reactive Oxygen Species in Presence of Alternaria solani

Resistance gene analogs (RGAs) comprising NBS-LRR gene family members are considered prominent candidates in the development of disease-resistant genotypes. NBS-LRR gene family comprised a very large number of genes; therefore, members of one subfamily TIR-NBS-LRR (TNL) are identified in the present study from Solanum tuberosum genome, followed by their bioinformatics characterization. The study identified a total of 44 genes encoding 60 TNL transcripts with two prominent clusters at chromosome 1 and chromosome 11. Expression analysis of 14 TNL genes after Alternaria solani infection at 1, 2, 3, 5, and 7 days post inoculation in two disease-tolerant varieties, Kufri Jyoti and Kufri Pukhraj, and one relatively susceptible variety, Kufri Chandramukhi, showed differential expression of many genes including a high expression (>15-fold) of StTNLC6G2T1 and StTNLC11G9T1. Functional characterization of one such gene, StTNLC7G2, reveals involvement in the generation of reactive oxygen species under A. solani attack, implicating its putative role in plant defense via hypersensitive response.

Essential Oils as Biocontrol Agents of Early and Late Blight Diseases of Tomato under Greenhouse Conditions

Tomato production worldwide is usually restrained by various infections, among them mainly the late and early blight caused by Phytophthora infestans and Alternaria solani, respectively. Lately, there has been a growing concern over the use of synthetic fungicides on environmental and food safety, hence the need to explore other alternatives that are friendly to the user, the consumer, and the general environment. This research sought to test the potency of ginger, garlic, and Mexican marigold essential oils against the early and late blight diseases of tomato under greenhouse conditions. A synthetic fungicide (Ridomil Gold®) was used as a positive control while distilled water acted as a negative control. The extraction of essential oils was done by dry steam distillation and then mixed with tween twenty before being topped up with sterile distilled water. They were then used to spray tomato plants that had been inoculated with A. solani and P. infestans isolates under greenhouse conditions. The tomato plants were evaluated for growth, yield, and disease severity. The data obtained was subjected to ANOVA and separation of means was conducted using Student–Newman–Keul (SNK) test at 95% level of confidence. The three essential oils had a significant potency against the two diseases which is comparable to the synthetic fungicide. Marigold essential oil was also found to have a significant impact on the general growth of sprayed tomato plants. Essential oils of the three plants can be further explored as alternative products management of the two diseases.

Effect of biotherapic of Alternaria solani on the early blight of tomato-plant and the in vitro development of the fungus

Background: homeopathy is a means permitted in organic agriculture to control disease and plagues; biotherapics are a practical means for farmers to intervene on the health of plants in agro-ecological systems of production. Tomato-plants can be affected by several diseases, one of the most significant ones in Brazil is early blight, caused by fungus Alternaria solani, due to the damage it causes and its wide distribution in the country. Aims: to establish whether a biotherapic of A. solani may interfere on the in vitro development of the fungus and whether it affects the severity of early blight on tomato-plants in greenhouse. Methods: the effect of the biotherapic on the fungus was evaluated through the percentage of germinated spores under microscope and the growth of colonies in a culture medium. Treatments used were: biotherapic 26cH, 27cH, 28cH, 29cH and 30cH; sterilized distilled water; and diluted and agitated hydroalcoholic solution. The effect of the biotherapic on the development of disease was evaluated in 4 experiments in greenhouse. Plants were kept in vases and subjected to artificial inoculation of the fungus after the application of treatments. Evaluation of disease was carried out through diagrammatic scale. Results: no treatment affected the germination of spores or the development of fungus colonies in the culture medium. In the first test, treatment 26cH differed from water in Tukey’s test at 5% but did not differed from diluted and agitated hydroalcoholic solution. In the second test, treatments 27cH and 28cH showed significant difference from both water and hydroalcoholic solution with an average control of disease of 57% and 62% respectively. The other 2 tests did nor exhibit any significant effect. Conclusions: there was no direct effect of the biotherapic on the fungus, but there was an effect on the severity of the disease. Factors affecting the efficiency of the biotherapic must be better understood before it can be recommended to farmers for the management of early blight in tomato-plants.

Assessment of Antifungal Efficacy and Release Behavior of Fungicide-Loaded Chitosan-Carrageenan Nanoparticles against Phytopathogenic Fungi

Biopolymeric Chitosan-Carrageenan nanocomposites 66.6–231.82 nm in size containing the chemical fungicide mancozeb (nano CSCRG-M) were synthesized following a green chemistry approach. The physicochemical study of nanoparticles (NPs) was accomplished using a particle size analyzer, SEM and FTIR. TEM exhibited clover leaf-shaped nanoparticles (248.23 nm) with mancozeb on the inside and entrapped outside. Differential scanning calorimetry and TGA thermogravimetry exhibited the thermal behaviour of the nanoform. Nano CSCRG-1.5 at 1.5 ppm exhibited 83.1% inhibition against Alternaria solani in an in vitro study and performed as well as mancozeb (84.6%). Complete inhibition was exhibited in Sclerotinia sclerotiorum at 1.0 and 1.5 ppm with the nanoformulation. The in vivo disease control efficacy of mancozeb-loaded nanoparticles against A. solani in pathogenized plants was found to be relatively higher (79.4 ± 1.7) than that of commercial fungicide (76 ± 1.1%) in pot conditions. Nanomancozeb showed superior efficacy for plant growth parameters, such as germination percentage, root–shoot ratio and dry biomass. The nanoformulation showed higher cell viability compared to mancozeb in Vero cell cultures at 0.25 and 0.50 mg/mL in the resazurin assay. CSCRG-0.5 showed slow-release behavior up to 10 h. Thus, these green nano-based approaches may help combat soil and water pollution caused by harmful chemical pesticides.