Phytopathogenic Fungus Alternaria Alternata Research Articles

Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Cynara cardunculus L. var. scolymus L. Landrace "Carciofo Ortano" as a Source of Bioactive Compounds.

The preservation of agricultural biodiversity and socioeconomic development are relevant both to enhance domestic production and to support innovation. In the search for new biomolecules, we have focused on the "Carciofo Ortano" landrace, growth in the northern part of the Lazio region. Artichoke cultivation generates substantial by-products, including leaves, stems, and roots, which could serve as valuable sources of biomolecules and prebiotic dietary fiber. To valorize the leaf waste of the "Carciofo Ortano" landrace, a multidisciplinary approach was applied. Chemical analysis using HPLC-DAD identified mono-O- and di-O-caffeoylquinic acids and the sesquiterpene cynaropicrin in all artichoke leaf extracts. SPME-GC/MS analyses detected aliphatic alcohols in the fresh leaf samples. Antiproliferative and cytotoxic studies on cancer (SH-SY5Y, MCF-7, MDA) and normal (MCF-10A) human cell lines revealed that leaf extracts induced a selective dose and time-dependent biological effect. While showing slight activity against environmental bacterial strains, artichoke leaf extracts exhibited significant antifungal activity against the phytopathogenic fungus Alternaria alternata. Overall, the results highlight the potential of "Carciofo Ortano" cultivation by-products as a rich source of biomolecules with versatile applications in humans, animals, and the environment.

Curcumin: An innovative approach for postharvest control of Alternaria alternata induced black rot in cherry tomatoes

Curcumin, a natural bioactive compound derived from Curcuma longa, has been widely recognized for its antifungal properties. In this study, we investigated the effects of curcumin on the phytopathogenic fungus Alternaria alternata and its pathogenicity in cherry tomato fruit. The results demonstrated that curcumin treatment significantly inhibited mycelial growth and spore germination of A. alternata in a dose-dependent manner. Scanning electron microscopy revealed alterations in the morphology of A. alternata mycelia treated with curcumin. Furthermore, curcumin treatment led to an increase in malondialdehyde and hydrogen peroxide contents, indicating cell membrane damage in A. alternata. Moreover, curcumin exhibited a remarkable inhibitory effect on the incidence and lesion diameters of black rot caused by A. alternata in cherry tomato fruit. Gene expression analysis revealed upregulation of defense-related genes (POD, SOD, and CAT) in tomato fruit treated with curcumin. Additionally, curcumin treatment resulted in decreased activity of exocellular pathogenic enzymes (polygalacturonase, pectin lyase, and endo-1,4-β-d-glucanase) in A. alternata. Overall, our findings highlight the potential of curcumin as an effective antifungal agent against A. alternata, providing insights into its inhibitory mechanisms on mycelial growth, spore germination, and pathogenicity in cherry tomato fruit.

Antifungal Activity of Streptomyces hygroscopicus JY-22 against Alternaria alternata and Its Potential Application as a Biopesticide to Control Tobacco Brown Spot

Tobacco brown spot caused by Alternaria alternata is a devastating fungal disease endangering plant production. To effectively control brown spot disease, an antagonistic Actinomyces strain, Streptomyces hygroscopicus JY-22, was isolated from rhizospheric soil, which remarkably restrains the growth of phytopathogenic fungus Alternaria alternata. This antagonistic strain and its culture filtrate showed significant antifungal activity against Alternaria alternata. Firstly, the confrontation culture method of strains JY-22 and Alternaria alternata showed that Streptomyces hygroscopicus JY-22 had noticeable antifungal activity against Alternaria alternata, including inhibition of mycelial growth and mycelial morphological changes, compared with the control group. Furthermore, the culture filtrate of Streptomyces hygroscopicus JY-22 showed substantial inhibition of the mycelial growth and spore germination of Alternaria alternata in a dose-dependent manner. Additional studies revealed that these antifungal actions were mainly related to membrane-active mechanisms that increased membrane permeability and damaged the cell membrane, leading to changes in certain cytoplasmic properties, such as extracellular conductivity and ergosterol, MDA and soluble protein content. In detached-leaf and field experiments, foliar spraying with culture filtrate resulted in smaller lesions and a lower disease index than the control group. Taken together, these results suggest that Streptomyces hygroscopicus JY-22 and its culture filtrate have the potential to be a safe biopesticide for the bio-control of tobacco brown spot.

The Regulatory Hub of Siderophore Biosynthesis in the Phytopathogenic Fungus Alternaria alternata.

A GATA zinc finger-containing repressor (AaSreA) suppresses siderophore biosynthesis in the phytopathogenic fungus Alternaria alternata under iron-replete conditions. In this study, targeted gene deletion revealed two bZIP-containing transcription factors (AaHapX and AaAtf1) and three CCAAT-binding proteins (AaHapB, AaHapC, and AaHapE) that positively regulate gene expression in siderophore production. This is a novel phenotype regarding Atf1 and siderophore biosynthesis. Quantitative RT-PCR analyses revealed that only AaHapX and AaSreA were regulated by iron. AaSreA and AaHapX form a transcriptional feedback negative loop to regulate iron acquisition in response to the availability of environmental iron. Under iron-limited conditions, AaAtf1 enhanced the expression of AaNps6, thus playing a positive role in siderophore production. However, under nutrient-rich conditions, AaAtf1 plays a negative role in resistance to sugar-induced osmotic stress, and AaHapX plays a negative role in resistance to salt-induced osmotic stress. Virulence assays performed on detached citrus leaves revealed that AaHapX and AaAtf1 play no role in fungal pathogenicity. However, fungal strains carrying the AaHapB, AaHapC, or AaHapE deletion failed to incite necrotic lesions, likely due to severe growth deficiency. Our results revealed that siderophore biosynthesis and iron homeostasis are regulated by a well-organized network in A. alternata.

Histone Methylation Is Required for Virulence, Conidiation, and Multi-Stress Resistance of Alternaria alternata

Histone methylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). This study determined the function of 5 HMTs (AaDot1, AaHMT1, AaHnrnp, AaSet1, and AaSet2) and 1 HDMs (AaGhd2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. The vegetative growth, conidiation, and pathogenicity of ∆AaSet1 and ∆AaSet2 were severely inhibited indicating that AaSet1 and AaSet2 play critical roles in cell development in A. alternata. Multiple stresses analysis revealed that both AaSet1 and AaSet2 were involved in the adaptation to cell wall interference agents and osmotic stress. Meanwhile, ∆AaSet1 and ∆AaSet2 displayed serious vegetative growth defects in sole carbon source medium, indicating that AaSet1 and AaSet2 play an important role in carbon source utilization. In addition, ∆AaSet2 colony displayed white in color, while the wild-type colony was dark brown, indicating AaSet2 is an essential gene for melanin biosynthesis in A. alternata. AaSet2 was required for the resistance to oxidative stress. On the other hand, all of ∆AaDot1, ∆AaHMT1, and ∆AaGhd2 mutants displayed wild-type phenotype in vegetative growth, multi-stress resistance, pathogenicity, carbon source utilization, and melanin biosynthesis. To explore the regulatory mechanism of AaSet1 and AaSet2, RNA-seq of these mutants and wild-type strain was performed. Phenotypes mentioned above correlated well with the differentially expressed genes in ∆AaSet1 and ∆AaSet2 according to the KEGG and GO enrichment results. Overall, our study provides genetic evidence that defines the central role of HMTs and HDMs in the pathological and biological functions of A. alternata.

Homology modeling, docking and molecular dynamics studies of some secondary metabolites of actinomycetes as biocontrol agents against the 3HNR enzyme of the phytopathogenic fungus Alternaria alternata

Early blight of tomatoes is a common disease caused by the phytopathogenic fungi Alternaria, in particular the species A. alternata. This disease causes significant losses in the tomato harvest. The enzyme 1,3,8-trihydroxynaphthalene reductase (3HNR) is a key enzyme involved in the production of melanin, that plays a crucial role in the process of fungi invasion. This enzyme is the target of some chemical fungicides, but the problem of resistance against these molecules requires the search for new molecules that are both effective and environment-friendly. Actinomycetes represent an important source of secondary metabolites with antimicrobial activity. Thus, in this study 110 secondary metabolites of actinomycetes were subjected to an in silico screening of their antifungal activity as possible inhibitors of the 3HNR of A. alternata. For this reason, the 3D structure of this enzyme was modeled. Then, a molecular docking study of the secondary actinomycetal metabolites was carried out within the catalytic site of the enzyme. Indole-3-carboxylic acid, Streptokordin, 3-Phenylpropionic acid, Phenylacetate, and 8-Hydroxyquinoline have shown the most promising results with binding energies of −6.1 kcal/mol, −6.1 kcal/mol, −5.4 kcal/mol, −5.3 kcal/mol, and −5.0 kcal/mol, respectively. These metabolites have also shown satisfactory results for drug-likeness and ADMET analysis. The interaction stability of the Streptokordin, Indole-3-carboxylic acid, Phenylacetate, and 8-Hydroxyquinoline within the catalytic site of 3HNR was confirmed by the results of the MD simulation and MM-PBSA analyzes. With their favorable interactive and pharmacokinetic characteristics, these metabolites may be potential antifungal molecules against A. alternata, and good candidates for further studies. Communicated by Ramaswamy H. Sarma

Histone Acetyltransferases and Deacetylases Are Required for Virulence, Conidiation, DNA Damage Repair, and Multiple Stresses Resistance of Alternaria alternata.

Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants (ΔGcn5, ΔRTT109, ΔElp3, ΔSas3, ΔNat3, ΔHos2, and ΔRpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2). ΔGcn5, ΔSas3, and ΔHos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in ΔGcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, ΔRTT109, ΔGcn5, and ΔHos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in ΔGcn5 and ΔHos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.

Distinct and essential roles of bZIP transcription factors in the stress response and pathogenesis in Alternaria alternata

The ability to cope with environmental abiotic stress and biotic stress is crucial for the survival of plants and microorganisms, which enable them to occupy multiple niches in the environment. Previous studies have shown that transcription factors play crucial roles in regulating various biological processes including multiple stress tolerance and response in eukaryotes. This work identified multiple critical transcription factor genes, metabolic pathways and gene ontology (GO) terms related to abiotic stress response were broadly activated by analyzing the transcriptome of phytopathogenic fungus Alternaria alternata under metal ions stresses, oxidative stress, salt stresses, and host-pathogen interaction. We investigated the biological functions and regulatory roles of the bZIP transcriptional factor (TF) genes in the phytopathogenic fungus A.alternata by analyzing targeted gene disrupted mutants. Morphological analysis provides evidence that the bZIP transcription factors (Gcn4, MeaB, Atf1, the ER stress regulator Hac1, and the all development altered-1 gene Ada1) are required for morphogenesis as the colony morphology of these gene deletion mutants was significantly different from that of the wild-type. In addition, bZIPs are involved in the resistance to multiple stresses such as oxidative stress (Ada1, Yap1, MetR) and virulence (Hac1, MetR, Yap1, Ada1) at varying degrees. Transcriptome data demonstrated that the inactivation of bZIPs (Hac1, Atf1, Ada1 and Yap1) significantly affected many genes in multiple critical metabolism pathways and gene ontology (GO) terms. Moreover,the ΔHac1 mutants displayed reduced aerial hypha and are hypersensitivity to endoplasmic reticulum disruptors such as tunicamycin and dithiothreitol. Transcriptome analysis showed that inactivation of Hac1 significantly affected the proteasome process and its downstream unfolded protein binding, indicating that Hac1 participates in the endoplasmic reticulum stress response through the conserved unfolded protein response. Taken together, our findings reveal that bZIP transcription factors function as key regulators of fungal morphogenesis, abiotic stress response and pathogenesis, and expand our understanding of how microbial pathogens utilize these genes to deal with environmental stresses and achieve successful infection in the host plant.

Identification and genomic characterization of a novel polymycovirus from Alternaria alternata causing watermelon leaf blight.

A double-stranded RNA (dsRNA) mycovirus from the phytopathogenic fungus Alternaria alternata, which causes watermelon leaf blight, was characterized. The genome of this virus has eight dsRNA segments, ranging from 1039 bp to 2398 bp. DsRNAs 1-6 each contain a single large open reading frame (ORF), while dsRNAs 7 and 8 each dsRNA contain two ORFs. The RNA-dependent RNA polymerase (RdRp) encoded by dsRNA1 and the viral methyltransferase encoded by dsRNA3 share 97.6% and 98.9% amino acid sequence identity, respectively, with the corresponding proteins of Plasmopara viticola lesion associated polymycovirus 1. The dsRNA5-encoded proline-alanine-serine-rich protein shows 48.1% sequence identity to that of Beauveria bassiana polymycovirus 3. The proteins encoded on dsRNAs 2, 4, and 8 have 99.7%, 98.2%, and 65.1% sequence identity, respectively, to the corresponding proteins of a mycovirus identified in Alternaria sp. FA0703 (AltR1). The proteins encoded by dsRNAs 6 and 7 do not match any known proteins of mycoviruses. Phylogenetic analysis of the RdRp domain showed that the virus clustered with members of the family Polymycoviridae. Based on these characteristics, the mycovirus was identified as a polymycovirus and designated as "Alternaria alternata polymycovirus 1" (AaPmV1). This is the first report of a polymycovirus associated with A. alternata.

Effect of humic acid on the composition of osmolytes and lipids in a melanin-containing phytopathogenic fungus Alternaria alternata

Humic substances (HS) have a direct impact on living cells, causing a wide range of various biological effects, and stimulating or inhibiting fungal growth. Therefore, it is important to reveal cellular indicators that could indicate the nature and level of HS’ effects on living organisms. Paying attention to the important role of lipid and osmolytes in adaptation to stress, the aim of this work was to study the composition of osmolytes and lipids in the growth dynamics of the phytopathogenic melanin-containing fungus Alternaria alternata under the influence of humic acid (HA). HA was isolated from a commercial peat humic product. For cultivation, liquid Czapek medium with the addition of 200 mg/l HA (HA variant) was used, and with no HA for the control variant. The main osmolytes of the fungal mycelium were glucose, mannitol, and trehalose. Both in the control and in HA variants, a general pattern was observed during the growth process; the amount of glucose increased against the background of a decrease of mannitol. In the control, the amount of trehalose increased significantly by the stage of active melanin formation (day 4), while under the influence of HA, such an increase was not observed. Membrane lipids were represented by phospholipids, sphingolipids, and sterols. A characteristic feature of A. alternata was a high proportion of phosphatidic acids (PA) in the composition of membrane lipids. The amount and proportion of PA decreased during growth in the control, while under the influence of HA such decline was not observed. The influence of HA induced the increase in the amount of sterols (St) and phosphatidylcholines (PC) that were able to stabilise the membrane lipid bilayer. We can assume that general cellular response of A. alternata to HA influence, being expressed as a higher amount of PA, PC, St and a lower level of trehalose in comparison with the control, indicates the an absence of inhibition of growth processes of this phytopathogenic melanin-producing fungus. The effect of HA on the qualitative and quantitative composition of the osmolytes and membrane lipids of A. alternata may cause changes of virulence and stress resistance of this phytopathogen.

Aaprb1, a subtilsin-like protease, required for autophagy and virulence of the tangerine pathotype of Alternaria alternata

Subtilisin-like serine protease secreted by pathogenic fungi can facilitate the infection and acquisition of nutrients. Functions of subtilisin-like serine proteases in the phytopathogenic fungus Alternaria alternata remains unknown. In the current study, 15 subtilisin-like serine proteases were individually deleted in the citrus fungal pathogen A. alternata. Only one, designated AaPrb1, was found to be required for A. alternata pathogenesis. The AaPrb1 deficiency strain (ΔAaprb1) reduced growth, conidiation, the formation of aerial hyphae, protease production, and virulence on citrus leaves. However, biochemical analyses and bioassays revealed that ΔAaprb1 plays no role in the production of ACT toxin. Through Y2H assays, Aaprb1 was found to interact with Aapep4, a vacuole-localized proteinase A in A. alternata. Furthermore, silencing AaPep4 in A. alternata resulted in phenotypes similar with those of ΔAaprb1. Expression of AaPrb1 was found to be regulated by AaPep4. TEM showed that AaPrb1and AaPep4 were involved in the suppression of the degradation of autophagosomes. Deletion of the autophagy gene AaAtg8 in A. alternata decreased conidiation, the formation of aerial hyphae and pathogenicity similar to ΔAaprb1, implying that some phenotypes of ΔAaprb1 were due to the impairment of autophagy. Overall, this study expands our understanding of how A. alternata utilizes the subtilisin-like serine protease to achieve successful infection in the plant host.

The siderophore repressor SreA maintains growth, hydrogen peroxide resistance, and cell wall integrity in the phytopathogenic fungus Alternaria alternata

The siderophore-mediated iron uptake machinery is required by the tangerine pathotype of Alternaria alternata to colonize host plants. The present study reports the functions of the GATA-type transcription regulator SreA by analyzing loss- and gain-of-function mutants. The expression of sreA is transiently upregulated by excess iron. The sreA deficiency mutant (ΔsreA) shows severe growth defect but produces ACT toxin and incites necrotic lesions on citrus leaves as efficiently as wild type. SreA suppresses the expression of genes encoding polypeptides required for siderophore biosynthesis and transport under iron-replete conditions. Under iron-replete conditions, SreA impacts the expression of the genes encoding the NADPH oxidase complex involved in H2O2 production. SreA negatively impacts H2O2 resistance as ΔsreA increases resistance to H2O2. However, sreA deficiency has no effects on the expression of genes encoding several key factors (Yap1, Hog1, and Skn7) involved in oxidative stress resistance. ΔsreA increases resistance to calcofluor white and Congo red, which may suggest a role of SreA in the maintenance of cell wall integrity. Those are novel phenotypes associated with fungal sreA. Overall, our results indicate that SreA is required to protect fungal cells from cytotoxicity caused by excess iron. The results also highlight the regulatory functions of SreA and provide insights into the critical role of siderophore-mediated iron homeostasis in resistance to oxidative stress in A. alternata.

Coding the α-subunit of SNF1 kinase, Snf1 is required for the conidiogenesis and pathogenicity of the Alternaria alternata tangerine pathotype

To well cope with various external carbon sources, fungi have evolved an adaptive mechanism to overcome the adversity of carbon source deficiency. The sucrose non-fermenting (SNF1) protein kinase mainly mediates the utilization of non-fermentable carbon sources. In this study, we determined the function of Snf1, coding the α-subunit of SNF1 kinase, in the phytopathogenic fungus Alternaria alternata via analyzing the Snf1 deletion mutants (ΔAasnf1). Aasnf1 is required for growth, development of aerial mycelium, and conidiation. Results of pathogenicity test showed that ΔAasnf1 induced smaller lesions on detached citrus leaves. Moreover, in the carbon utilization assay, ΔAasnf1 showed growth inhibition on the minimal medium supplemented with polygalacturonic acid, sucrose or alcohol as the only carbon source. Compared to the wild type, ΔAasnf1 also exhibited stronger resistance to cell wall stressors of sodium dodecyl sulfate and congo red. In conclusion, Aasnf1 played important roles in the carbon utilization, vegetative growth, conidiation, cell wall functions and pathogenicity of A. alternata. This study is the first report on the functions of Aasnf1 and our results suggest that Snf1 is critical for the conidiogenesis and pathogenesis of the A. alternata tangerine pathotype.

Molecular and biological characterization of a novel botybirnavirus identified from a Pakistani isolate of Alternaria alternata

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

AbstractThe endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.

Ethylene production by Alternaria alternata and its association with virulence on inoculated grape berries

Ethylene has been shown to promote spore germination and hyphal growth in the phytopathogenic fungus Alternaria alternata. However, little is known about the ethylene biosynthetic pathway in this fungus. In the present study, the ethylene biosynthetic pathway in A. alternata was investigated to explore ethylene-associated virulence of this fungus. The strain A0 of A. alternata did not produce ethylene on basal medium with different possible precursors or intermediates for ethylene biosynthesis (glutamate, aspartate, 2-oxoglutarate and 1-aminocyclopropano −1-carboxylic acid). However, ethylene production was observed when methionine was added as a precursor to the medium and was further promoted by continuous light illumination. Furthermore, addition of 2-keto-4-methylthiobutyric acid (KMBA) promoted ethylene production in the absence of methionine, indicating that the KMBA pathway was mainly responsible for ethylene biosynthesis in this fungus. The strain A0 was inoculated into grape berries to examine the effect of ethylene production on its virulence (as assessed by lesion formation at the inoculation site). The results indicated that higher ethylene production caused larger lesion formation. Similar results were also obtained when isolates of A. alternata, obtained from infected grapes, were inoculated. Thus, the present study thus demonstrated that A. alternata produces ethylene via the KMBA pathway and utilizes it for enhanced virulence expression during infection.

A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the ‘two component’ histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

Copper tolerance, protein and catalytic activity in phytopathogenic fungus Alternaria alternata

<div> <p><em>In vitro </em>tolerance of soil borne phytopathogenic fungus i.e. <em>Alternaria alternata </em>in terms of growth, physiology and metal uptake capacity against different doses of copper [Cu(II)] metal was assessed. In preliminary growth experiments, nitrate, chloride and sulphate of Cu(II) were amended in seven different concentrations viz. 25, 50, 75, 80, 85, 90 and 100 ppm in nutritive agar medium along with fungus inoculum. Amongst three salts, the maximum inhibition in radial growth of the fungus was observed with increasing concentrations of copper salts in order of: CuSO<sub>4</sub>> CuNO<sub>3</sub>> CuCl<sub>2</sub>. Further trials were conducted in nutritive broth with four different concentrations (25, 50, 75 and 100 ppm) of copper sulphate to assess growth inhibition, metal accumulation, protein and catalase activity in the fungus biomass. It was found that fungus dry biomass was significantly declined by 70-99% along with net metal accumulation of 40% with increasing metal concentrations. Total protein contents and catalase activity was considerably increased up to 2-5 and 2-3 folds, respectively with increase in metal concentration from 25-100 ppm and incubation period from 48, 72 and 96 hours over control. Present study concludes that <em>A. alternata </em>was able to tolerate up to 90 ppm of copper, therefore it is imperative to consider fungus metal tolerance range during fungicide formulations.</p> </div> <p> </p>

FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants

In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth.