Early Blight Pathogen Research Articles

CRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions.

Potato is the third most important food crop, but cultivation is challenged by numerous diseases and adverse abiotic conditions. To combat diseases, frequent fungicide application is common. Knocking out susceptibility genes by genome editing could be a durable option to increase resistance. DMR6 has been described as a susceptibility gene in several crops, based on data that indicates increased resistance upon interruption of the gene function. In potato, Stdmr6-1 mutants have been described to have increased resistance against the late blight pathogen Phytophthora infestans in controlled conditions. Here, we present field evaluations of CRISPR/Cas9 mutants, in a location with a complex population of P. infestans, during four consecutive years that indicate increased resistance to late blight without any trade-off in terms of yield penalty or tuber quality. Furthermore, studies of potato tubers from the field trials indicated increased resistance to common scab, and the mutant lines exhibit increased resistance to early blight pathogen Alternaria solani in controlled conditions. Early blight and common scab are problematic targets in potato resistance breeding, as resistance genes are very scarce. The described broad-spectrum resistance of Stdmr6-1 mutants may further extend to some abiotic stress conditions. In controlled experiments of either drought simulation or salinity, Stdmr6-1 mutant plants are less affected than the background cultivar. Together, these results demonstrate the prospect of the Stdmr6-1 mutants as a useful tool in future sustainable potato cultivation without any apparent trade-offs.

The Effect of Rhizospheric Fungal Species of Tomato (Solanum lycopersicum L.), on In-Vitro Growth of the Tomato Early Blight Pathogen

Tomato (Solanum lycopersicum L.) is a globally consumed crop, susceptible to various biotic and abiotic stresses. Among biotic stresses, plant diseases, particularly Early Blight caused by Alternaria spp. leads to heavy losses in the field and post-harvest stages. The conventional use of synthetic agrochemicals to control these diseases poses environmental risks, underscoring the need for sustainable alternatives. Hence, this study aimed to assess the potential of rhizospheric fungi in controlling Alternaria spp. Infected tomato leaves, stem parts, and fruits were collected from commercial cultivations in four agro-ecological regions in Sri Lanka (two from mid-country in the wet zone, and two from the intermediate zone). All samples were washed using running tap water, and 1cm2 sections, including the edges of lesions, were cut, surface sterilized, placed on potato dextrose agar (PDA), and incubated at room temperature until colonies appeared. Fungal colonies that emerged from lesions, were sub-cultured onto fresh PDA, and pure cultures were obtained using the hyphal-tip purification method. Alternaria isolates were identified morphologically and confirmed the pathogenicity by Koch’s postulate method. Species-level identification involved genomic DNA extraction and sequencing ITS regions. The soil dilution plate technique was used to isolate rhizospheric fungi from two agroecological regions. Composite samples (10 g) of soil attached to the roots of 5 uprooted healthy plants, from each field were used. Soil suspensions were inoculated onto PDA using the spread-plate method and incubated at room temperature for 5 days. A total of 27 isolates were obtained and morphologically identified up to the genus level after preparing pure cultures. The dual-culture plate technique was used to assay percentage inhibition, and according to Tukey’s pairwise comparison, Trichoderma sp. and Purpureocillium lilacinum exhibited significantly high (p≤0.05) inhibition of Alternaria colony growth, (68.25±1.18% and 64.35±3.31% respectively). Microscopic observations of inhibition zones between Alternaria and antagonistic rhizospheric fungal colonies, including Trichoderma sp. and P. lilacinum showed the presence of mycoparasitism-related structures, such as haustoria, coils, loops, and knobs. Qualitative assay for fungal chitinase enzyme production demonstrated high activities in P. lilacinum, Trichoderma sp. and Acremonium sp., whereas in a quantitative assay for fungal glucanase enzyme, testing the ability of fungal culture filtrates to release 1µg of glucose per minute, from Beta-glucanase solution, P. lilacinum, Trichoderma sp., and Paecilomyces sp. showed significantly high (p≤0.05) activities (105.14± 3.62, 100.11±4.09 and 99.83 ±6.44 μg glucose/L respectively). In conclusion, this study highlights the potential of rhizospheric fungi like P. lilacinum and Trichoderma sp. as effective biocontrol agents against Alternaria spp. and these findings lay the groundwork for future applications in enhancing tomato crop resilience against pathogens and promoting sustainable and eco-friendly agricultural practices.
 
 Keywords: Rhizospheric fungi, Mycoparasitism, ITS region, Chitinase enzyme, Glucanase enzyme

Positive selection and functional diversification of transcription factor Cmr1 homologs in Alternaria

Transcription factor Cmr1 (Colletotrichum melanin regulation 1) and its homologs in several plant fungal pathogens are the regulators of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis pathway and have evolved functional diversification in morphology and pathogenicity. The fungal genus Alternaria comprises the group of “black fungi” that are rich in DHN-melanin in the primary cell wall and septa of the conidia. Some Alternaria species cause many economically important plant diseases worldwide. However, the evolution and function of Cmr1 homologs in Alternaria remain poorly understood. Here, we identified a total of forty-two Cmr1 homologs from forty-two Alternaria spp. and all contained one additional diverse fungal specific transcription factor motif. Phylogenetic analysis indicated the division of these homologs into five major clades and three branches. Dated phylogeny showed the A and D clades diverged latest and earliest, respectively. Molecular evolutionary analyses revealed that three amino acid sites of Cmr1 homologs in Alternaria were the targets of positive selection. Asmr1, the homolog of Cmr1 in the potato early blight pathogen, Alternaria solani was amplified and displayed the sequence conservation at the amino acid level in different A. solani isolates. Asmr1 was further confirmed to have the transcriptional activation activity and was upregulated during the early stage of potato infection. Deletion of asmr1 led to the decreased melanin content and pathogenicity, deformed conidial morphology, and responses to cell wall and fungicide stresses in A. solani. These results suggest positive selection and functional divergence have played a role in the evolution of Cmr1 homologs in Alternaria.Key points• Cmr1 homologs were under positive selection in Alternaria species• Asmr1 is a functional transcription factor, involved in spore development, melanin biosynthesis, pathogenicity, and responses to cell wall and fungicide stresses in A. solani• Cmr1 might be used as a potential taxonomic marker of the genus Alternaria

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC–MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-β-d-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Microbial consortium mediated acceleration of the defense response in potato against Alternaria solani through prodigious inflation in phenylpropanoid derivatives and redox homeostasis

The present study was carried out in a pot experiment to examine the bioefficacy of three biocontrol agents, viz., Trichoderma viride, Bacillus subtilis, and Pseudomonas fluorescens, either alone or in consortium, on plant growth promotion and activation of defense responses in potato against the early blight pathogen Alternaria solani. The results demonstrate significant enhancement in growth parameters in plants bioprimed with the triple-microbe consortium compared to other treatments. In potato, the disease incidence percentage was significantly reduced in plants treated with the triple-microbe consortium compared to untreated control plants challenged with A. solani. Potato tubers treated with the consortium and challenged with pathogen showed significant activation of defense-related enzymes such as peroxidase (PO) at 96 h after pathogen inoculation (hapi) while, both polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) at 72 hapi, compared to the individual and dual microbial consortia-treated plants. The expression of antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) and the accumulation of pathogenesis-related proteins such as chitinase and β-1,3-glucanase were observed to be highest at 72 hapi in the triple microbe consortium as compared to other treatments. HPLC analysis revealed significant induction in polyphenolic compounds in triple-consortium bioprimed plants compared to the control at 72 hapi. Histochemical analysis of hydrogen peroxide (H2O2) clearly showed maximum accumulation of H2O2 in pathogen-inoculated control plants, while the lowest was observed in triple-microbe consortium at 72 hapi. The findings of this study suggest that biopriming with a microbial consortium improved plant growth and triggered defense responses against A. solani through the induction of systemic resistance via modulation of the phenylpropanoid pathway and antioxidative network.

Bacillus Amyloliquefaciens IKMM and Zinc Nanoparticles as Biocontrol Candidate Induce the Systemic Resistance by Producing Antioxidants in Tomato Plants Challenged with Early Blight Pathogen

Bacillus Amyloliquefaciens IKMM and Zinc Nanoparticles as Biocontrol Candidate Induce the Systemic Resistance by Producing Antioxidants in Tomato Plants Challenged with Early Blight Pathogen

Use of Trichoderma culture filtrates as a sustainable approach to mitigate early blight disease of tomato and their influence on plant biomarkers and antioxidants production.

Alternaria solani is a challenging pathogen in the tomato crop globally. Chemical control is a rapid approach, but emerging fungicide resistance has become a severe threat. The present study investigates the use of culture filtrates (CFs) of three species of Trichoderma spp. to control this disease. Highly virulent A. solani strain and three Trichoderma fungal strains viz., T. harzianum (Accession No: MW590687), T. atroviride (Accession No: MW590689) and T. longibrachiatum (Accession No: MW590688) previously isolated by authors were used in this study. The efficacy of culture filtrates (CFs) to mitigate early blight disease were tested under greenhouse and field conditions, experiments were conducted in different seasons of 2020 using a tomato variety "doucen". The CFs of T. harzianum, T. longibrachiatum, and T. atroviride significantly inhibited the in vitro mycelial growth of A. solani (62.5%, 48.73%, and 57.82%, respectively, followed by control 100%). In the GC-MS analysis of Trichoderma CF volatile compounds viz., harzianic acid (61.86%) in T. harzianum, linoleic acid (70.02%) in T. atroviride, and hydroxymethylfurfural (68.08%) in the CFs of T. longibrachiatum, were abundantly present. Foliar application of CFs in the greenhouse considerably reduced the disease severity (%) in all treatments, viz., T. harzianum (18.03%), T. longibrachiatum (31.91%), and T. atroviride (23.33%), followed by infected control (86.91%), and positively affected the plant biomarkers. In the greenhouse, the plants treated with CFs demonstrated higher flavonoids after 6 days of inoculation, whereas phenolic compounds increased after 2 days. The CF-treated plants demonstrated higher antioxidant enzymes, i.e., phenylalanine ammonia-lyase (PAL) and peroxidase (POD), after 4 days, whereas polyphenol oxidase (PPO) was higher after 6 days of inoculation, followed by healthy and infected controls. In open field conditions, disease severity in CF-treated plants was reduced in both seasons as compared to naturally infected plants, whereas CF-treated plants exhibited a higher fruit yield than controls. The present results conclude that CFs can be a potential biocontrol candidate and a promising alternative to the early blight pathogen for sustainable production.

Inhibition of the Growth and Development of Potato Early Blight Pathogen (Alternaria solani) by Combining Penicillium chrysogenum VKM F-4876D with Some Strobilurin-, Triazole-, and Phenylpyrrole-Based Fungicides

The biological control of fungal crop diseases based on the use of micro-organisms or their metabolites is a promising environmentally friendly alternative to common fungicide-based technologies. Penicillium fungi which synthesize various biologically active compounds with a wide range of antimicrobial activity are of special interest as potential producers of antifungal biopreparations. The purpose of this study was the evaluation of the antifungal activity of a dry biomass of the Penicillium chrysogenum VKPM F-4876D strain (DMP), both individually and combined with commercial azoxystrobin-, fludioxonil-, difenoconazole-, and tebuconazole-based fungicides commonly used to control early blight, towards Alternaria solani, an infectious agent of this potato disease. The study was performed under in vitro (cultivation on solid and liquid media) and in vivo (detached potato leaves) conditions. The cultivation of A. solani on agarized medium supplemented with 5 g/L of DMP caused significant morphological changes in pathogen colonies, whereas 7.5 g/L of DMP resulted in complete suppression of pathogen development. DMP addition to the liquid culture of A. solani significantly increased electrolyte release, i.e., impaired the cell membrane integrity in the pathogen. Combining DMP (1–2 g/L) with fungicides at a dose range of 0.01–10 mg/L resulted in significant in vitro growth inhibition of A. solani. The best result was obtained for the combination of DMP (1 g/L) with fludioxonil (1–5 mg/L) or tebuconazole (10 mg/L): a significant and prolonged antifungal effect (96–97% growth inhibition) was observed during the whole 14-day period of observation. The treatment of detached potato leaves with 1 g/L of DMP prior to artificial infection with A. solani resulted in a four-fold increase in the number of uninfected leaves and a two-fold reduction in the number of severely infected leaves compared to the control. These results demonstrate the possibility to develop an efficient environmentally friendly approach to manage potato early blight and provide prerequisites for the reduction in pesticide load on agrobiocoenoses, thus contributing to the restoration of ecological balance and the prevention of the emergence and spread of resistant pathogen strains.

Multiomic approaches reveal novel lineage-specific effectors in the potato and tomato early blight pathogen Alternaria solani

The effectome of the necrotrophic fungal pathogen, Alternaria solani, was determined using multiomics. In total, 238 effector candidates were predicted from the A. solani genome, and apoplastic effectors constitute most of the total candidate effector proteins (AsCEPs). Comparative genomics revealed two main groups of AsCEPs: lineage-specific and conserved effectors. RNA-Seq analysis revealed that the most highly expressed genes encoding AsCEPs were enriched with lineage-specific forms. Two lineage-specific effector genes, AsCEP19 and AsCEP20, were found to form a ‘head-to-head’ gene pair located near an AT-rich region on the chromosome. To date, AsCEP19 and AsCEP20 have been found only in a few fungal species. Phylogenetic inference revealed that AsCEP19 and AsCEP20 were likely acquired by the common ancestor of A. solani and A. tomatophila via horizontal gene transfer, probably mediated by long terminal repeat retrotransposon. RT-qPCR analysis showed that AsCEP19 and AsCEP20 are tightly coexpressed in a host-specific manner and that they are upregulated at advanced stages of A. solani infection only in solanaceous hosts. Transient expression of AsCEP19 and AsCEP20 in Nicotiana benthamiana plants showed that these effectors could promote Phytophthora infestans infection. AsCEP19 and AsCEP20 were required for the full virulence of A. solani on host potato, because deletion of this gene pair significantly reduced the size of necrotic lesions on potato leaves. Transient expression of AsCEP20 could elicit plant cell death depending on the presence of its signal peptide, indicating that AsCEP20 is a necrosis-inducing apoplastic effector with the mature form localized specifically in chloroplasts. Our work provides a better understanding of the function and evolution of necrotrophic fungal effectors, and helps explain the high aggressiveness of A. solani against solanaceous crops.

Efficacy of native Trichoderma asperellum in managing Alternaria solani causing early blight of Solanum lycopersicum Mill

The biocontrol efficacy of Trichoderma asperellum strain M23 (bioagent) was tested against early blight pathogen (Alternaria solani strain NO4) of tomato. These fungi were co-inoculated on tomato plants (variety UC82B and Ibadan local) at different spore concentrations, viz., 1.0 × 105, 1.0 × 107 and 1.0 × 109 while untreated plant served as control. The effect of these treatments was studied based on growth parameters, biomass and symptoms observed on disease incidence scale of 1–7. The pathogen exerted varying effects on the tomato plants as Ibadan local was more susceptible compared to UC82B. The bioagent M23 exhibited significant growth stimulatory effect (p < 0.05) on the two varieties of tomato. The antagonistic effect of M23 against NO4 was confirmed on the growth, biomass accumulation (phytomass) and fruit weight of tomato plants co-inoculated with the pathogen and the antagonist. This study demonstrated the potentials of Trichoderma asperellum not only as biocontrol agent against early blight of tomatoes but also as growth stimulatory agent for the crop.

EVALUATION OF IN VITRO EFFECTIVENESS OF SELECTED FUNGICIDES FOR THE MANAGEMENT OF EARLY BLIGHT OF TOMATO CAUSED BY ALTERNARIA SOLANI IN ETHIOPIA

Tomato is one of the most popular vegetables grown throughout the world in general and in Ethiopia in particular. Production of the crop is constrained by different diseases and the disease caused by the early blight pathogen, Alternaria solani, is among the major ones. Early blight has been considered as commonly occurring and causing a significant economic impact on the yield and quality of the crops. Major tomato diseases in general and early blight, in particular, could be best managed by integrating a number of control practices and the application of fungicides. The application of fungicides is the most convenient and predominant way for disease control in the absence of resistant varieties. The present study was conducted to evaluate the efficacy of some selected fungicides with the objectives to assess in vitro efficacy of fungicides on the growth and sensitivity of pathogens, in vitro tolerance of pathogens to fungicides, and their effectiveness as potential foliar sprays against A. solani. A. solani was isolated from tomatoes showing typical early blight symptoms. Five fungicides viz. URGI 75% WP, Carbancholar 50%, Orozole 25 EC, Agro Laxyl MZ 63.5 WP, and Mancodex Super 72 WP were tested against the pathogen using poisoned food technique. The highest percentage reduction in the growth of A. solani was recorded with Orozole 25 EC followed by Agro Laxyl MZ 63.5 WP resulting in 100% and 56.5% inhibition in mycelial growth respectively. On the other hand, Carbancholar 50% was found the least effective in reducing the mycelial growth of A. Solani. Fungicides that showed better in vitro efficacy could be further tested for their efficacies under field conditions and can be verified for economic validity. The study also showed the potential effectiveness of fungicides for the management of early blight on tomatoes.

New Insight in the Occurrence of Early Blight Disease on Potato Reveals High Distribution of Alternaria solani and Alternaria protenta in Serbia.

Early blight is an economically important disease of potato worldwide. Understanding which fungal pathogens are the causal agents of early blight and their distribution on the same host is essential to finding the best strategy for the control of this disease. Previous studies have shown that Alternaria solani is the main early blight pathogen parasitizing potato. Here, we analyzed genetic and phenotypic diversity in isolates of Alternaria spp. covering all potato production areas in Serbia. We showed that the four species of Alternaria were found in areas with different distributions of the species. The occurrence of Alternaria spp. was studied by analyzing isolates from symptomatic potato leaves during multiyear sampling. In addition to Alternaria solani, we detected three more large-spored species identified as A. linariae (syn. A. tomatophila), A. protenta, and A. grandis that were involved in early blight disease on naturally infected potato leaves in Serbia. Differentiation of species was supported by phylogeny obtained from the DNA sequences of the GAPDH, calmodulin and Rpb2 genes. Our findings present a new perspective into the population structure of large-spored Alternaria species associated with early blight disease. Within the groups of large-spored Alternaria present in Serbia, evidence of A. protenta at high frequency reveals new insight into the contribution of Alternaria species in early blight disease. This work opens new perspectives for early blight management, while the distribution of different species on the same host suggests that the etiology of disease could depend on crop organization and the presence of other Alternaria hosts in close proximity to potato plants.

Plant physiology, microbial community, and risks of multiple fungal diseases along a soil nitrogen gradient

Nitrogen (N) is an essential element for plant growth and development. N levels in soil may also impact plant disease occurrence. However, the physiological and microbial mechanisms between N levels in soil and plant disease occurrence are not quite clear at present. In this study, we examined the impact of seven urea levels (0 to 800 mg kg−1 soil) on the physiology of tomato (Lycopersicon esculentum) and fungal disease occurrence. Our results showed that the disease incidence and index caused by a tomato early blight pathogen (Alternaria alternata) increased with increasing N levels. The disease index and percentage of disease incidence were positively correlated with N content, indole-3-acetic acid (IAA) and salicylic acid (SA) in plants, but negatively correlated with fungal community diversity. In addition to pathogens (A. alternata) that cause known early blight, 39 other fungal taxa were also identified as plant pathogens in tomato roots, leaves, and soil, the dominant putative pathogens included Ceratobasidiaceae sp., Fusarium oxysporum, Macrophomina phaseolina, Nectriaceae sp., Podosphaera fusca and Trichoderma viride. N levels affected the distribution and dynamics of the fungal pathogen community, such as the abundance of Fusarium oxysporum increased by 33% on roots from days 25 to 35. Our results demonstrated that plants undergo complex disease risk from different pathogens under different N levels, highlighting a need for proper N management, and integration of nutrient management as a disease control approach for sustainable agricultural production.

Whole-genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani.

Early blight of potato is caused by the fungal pathogenAlternaria solaniand is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole-genome sequencing for 48A. solaniisolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non-admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity ofA. solanion a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

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.

Impact of foliar application of fungicides on tomato leaf fungal community structure revealed by metagenomic analysis.

Fungicides are commonly used to manage plant pathogens. However, little is known about their effects on the non-target fungal communities that inhabit inside and outside the plant. These fungicides may have adverse effects on beneficial microbial communities with possible consequences for plant health and productivity. Hence, a metagenomic approach, based on the ITS2 region of fungal rDNA, was used to study the impact of foliar application of two fungicides (propineb and iprodione + carbendazim) on non-target tomato leaf fungal communities, in the context of early blight disease management. Metagenomic analysis revealed that the richness and diversity of tomato leaf fungal populations were adversely affected by the chemical treatments tested. Among the two fungicides, propineb (contact fungicide) imparted less non-targeted microorganisms than iprodione + carbendazim (systemic fungicide). In addition, all samples showed association of pathogenic genera Cladosporium, Corynespora, Pseudocercospora along with early blight pathogen Alternaria on tomato leaves that otherwise were undetected. Metagenomic studies also revealed a new mode of action for fungicides and bioagents besides their direct effect that is shifting the microbial community structure so that it provides greater resistance against the pathogen.

In-vitro Evaluation of Fungicide Sensitivity of Tomato Leaf Blight Pathogens

Tomato early and late blight diseases caused by Alternaria solani and Phytophthora infestans respectively, are constraints to tomato production globally. Conventional use of commercial synthetic fungicides in management of tomato blight disease has become a key input for tomato production among farmers in Tharaka Nithi County, Kenya. Indiscriminate use and application of sub-lethal doses of fungicides negate disease management efforts, and may lead to gradual fungicide resistance. Yet, local tomato farmers have continued to use the fungicides without periodic evaluation of their effectiveness. This study was carried out in-vitro to evaluate the efficacy of six synthetic commercial fungicides used by farmers around River Ruguti, against two tomato leaf blight pathogens; Alternaria solani and Phytophthora infestans. The poison food method was used to evaluate fungicides known by trade names and application levels; Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1), Mancozeb 640 g/kg + Metalaxyl 80 g/kg, Mancozeb, Propineb700 g/kg + Cymoxanil 60 g/kg, Carbendazim and Triticonazole at different concentration (25%, 50% and 75%). The in-vitro plate experiment was laid out in a Complete Randomized Design with 3 replicates, and data on mycelia growth inhibition analyzed through General Linear Model (α=.05) and significant means separated using Least significant difference (LSD) using Scientific Analysis System version 9.4. All the tested fungicides significantly (p≤ 0.05) inhibited mycelial growth of tested pathogen. Percentage inhibition for early blight pathogen (Alternaria solani) was 80.42% compared to late blight pathogen Phytophthora infestans at 69.51%. Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1) and Propineb700 g/kg + Cymoxanil 60 g/kg recorded higher per cent inhibition of mycelia growth of 92.4% and 89.71% respectively. Carbendazim recorded lower per cent inhibition of 39.15%. Mycelia growth inhibition increased with an increase in fungicide concentration. Lower inhibition of 71.78% was observed at 25% concentration as compared to 50% and 75% with 76.77% and 76.36% respectively. Fungicides screened varied in mycelia inhibition against P. infestans and A. solani isolates with Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1) and Propineb700g/kg +Cymoxanil 60g/kg giving significantly (p≤ 0.05) better inhibition while Carbendazim had the lowest inhibition effect. Increased fungicide concentration effectively inhibited mycelia growth. Thus higher concentration of fungicide application is recommended in cases where there is low efficacy of fungicides.

Efficacy of some conventional fungicides and essential oils against tomato early blight disease

Fungicidal activity of nine selected conventional fungicides, identified five plant oils and their combined effect against A. solani, the causal pathogen of early blight on tomato. GC-MS analysis of the tested oils indicated that terpene hydrocarbons (97%) with cinnamaldehyde as the major compound; sulfur compounds; fatty acids and organo-sulfur-containing compounds were the main components in cinnamon, garlic, mustard and onion oils, respectively. In vitro, Speedcide® (difenoconazole) and Cabrioduo® (Dimethomorph + Pyraclostrobin) were the most effective against the A. solani fungus with EC50 values of 94.6 and 88.6 ppm respectively. Toledo® (Tebuconzole) achieved EC50 value of 631.2 ppm. Roxyl-M® was significantly more effective than Roxyl–plus® or Remik®. Speedcide® alone or mixed with mandipropamid or azoxystrobin in Revus-Top® or Cruze® were the lowest effective in vitro. Garlic and mustard oils were more effective in liquid media than solid one. Cinnamon, onion, garlic and bitter almond oils caused moderate fungi-toxicity against early blight pathogen and could be implemented in the IPM program. Lower EC50 values were obtained in case of combinations of fungicides with oils than separate treatments. Effect of the active conventional fungicides and plant oils on disease indices, relative disease control, chlorophyll content, sugar contents and poly phenol oxidase in tomato plants was also studied.

Fungal Endophytes as Growth Promoters and Inducers of Resistance in Tomato (Lycopersicon esculentum Mill.) against Alternaria solani

Bio control strategies are gaining importance presently due to adverse effects caused by chemical pesticides on the environment. During our study, a total of 78 fungal endophytes were isolated from five medicinally important plants and evaluated for their efficacy in inducing resistance against the early blight pathogen caused by Alternaria solani. Among the isolates evaluated, only five endophytes were able to antagonize A. solani with a maximum inhibition of 58.53% offered by ENSM-08. All the antagonistic endophytes were molecularly identified based on ITS1 and ITS4 regions and sequence analysis was submitted to GenBank, NCBI to acquire accession numbers. In addition, seed treatment with the conidial suspension of the antagonistic fungal endophytes were able to enhance seed, vegetative and reproductive growth parameters in tomatoes with the highest enhancement observed upon the application of Pestalotiopsis microspora- ENSM-08. Also, seed treatments with select endophytes were also able to induce resistance in tomato plants against the early blight pathogen with a maximum protection of 70.25% observed in P. microspora- ENSM-08 treated plants. The results of the study validate the application of fungal endophytes as inoculants for sustainable agriculture.