Fungal Pathogen Alternaria Alternata Research Articles

Greenhouses represent an important evolutionary niche for Alternaria alternata.

Greenhouses have become increasingly important for food production and food security. However, our understanding of how greenhouses may contribute to genetic variations in soil microbial populations is very limited. In this study, we obtained and analyzed soil populations of the cosmopolitan fungal pathogen Alternaria alternata in seven greenhouses in Shijiazhuang, northeast China. Our analyses revealed high proportions of isolates being resistant to agricultural triazole fungicides and medical triazole drugs, including cross-resistance to both groups of triazoles. In addition, we found that greenhouse populations of A. alternata located within a few kilometers showed similar levels of genetic differentiation as those separated by over 2,000 km between northeast and southwest China. Our study suggests that greenhouse populations of this and potentially other fungal pathogens represent an important ecological niche and an emerging threat to food security and human health.

The microRNA miR482 regulates NBS-LRR genes in response to ALT1 infection in apple

Plants are frequently attacked by a variety of pathogens and thus have evolved a series of defense mechanisms, one important mechanism is resistance gene (R gene)-mediated disease resistance, but its expression is tightly regulated. NBS-LRR genes are the largest gene family of R genes. microRNAs (miRNAs) target to a number of NBS-LRR genes and trigger the production of phased small interfering RNAs (phasiRNAs) from these transcripts. phasiRNAs cis or trans regulate NBS-LRR genes, which can result in the repression of R gene expression. In this study, we screened for upregulated miR482 in the susceptible apple cultivar 'Golden Delicious' (GD) after inoculation with the fungal pathogen Alternaria alternata f. sp. mali (ALT1). Additionally, through combined degradome sequencing, we identified a gene targeted by miR482, named MdTNL1, a gene encoding a TIR-NBS-LRR (Toll/interleukin1 receptor-nucleotide binding site-leucine-rich repeat) protein. This gene exhibited a significant down-regulation post ALT1 inoculation, suggesting an impact on gene expression mediated by miRNA regulation. miR482 could cleave MdTNL1 and generate phasiRNAs at the cleavage site. We found that overexpression of miR482 inhibited the expression of MdTNL1 and thus reduced the disease resistance of GD, while silencing of miR482 increased the expression of MdTNL1 and thus improved the disease resistance of GD. This work elucidates key mechanisms underlying the immune response to Alternaria infection in apple. Identification of the resistance genes involved will enable molecular breeding for prevention and control of Alternaria leaf spot disease in this important fruit crop.

NaWRKY70 is a key regulator of Nicotiana attenuata resistance to Alternaria alternata through regulation of phytohormones and phytoalexins biosynthesis.

Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood. We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus-induced gene silencing. Silencing NaWRKY70 dramatically reduced both basal and A. alternata-induced jasmonoyl-isoleucine (JA-Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W-boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1-like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4-like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2-like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis. Our results highlight the key role of NaWRKY70 in controlling both JA-Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata.

Sodium nitroprusside (SNP) treatment increases the postharvest resistance of apple fruit to Alternaria alternata by enhancing antioxidant enzyme activity

Black spot disease, caused by the fungal pathogen Alternaria alternata, commonly infects apple (Malus domestica) during postharvest storage and transportation, diminishing fruit quality, contaminating the fruit with toxins, and causing significant economic losses. Here, we investigated the effect of 0.1 mmol L−1 sodium nitroprusside (SNP), a nitric oxide donor, on lesion diameter, fruit quality, and reactive oxygen species (ROS) metabolism in A. alternata-inoculated apples. SNP not only effectively decreased lesion diameter but also maintained fruit quality and cell membrane integrity. Additionally, SNP enhanced antioxidant enzyme activity and genes encoding of peroxidase (POD), glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), and decreased hydrogen peroxide (H2O2) and superoxide anion (O2•−) production, malondialdehyde (MDA) concentration, and membrane permeability. Collectively, these results demonstrate that SNP inhibits ROS accumulation and increases antioxidant enzyme activity, and that the mechanism of SNP-induced resistance is related to the regulation of ROS metabolism.

Study on the control potential of Alternaria alternata against four species of weeds in agricultural fields on the Tibetan Plateau

ABSTRACT The aim of this study was to investigate the potential and pathogenic mechanism of the pathogenic fungus Alternaria alternata strain GD-011-B against four major agricultural weeds on the Tibetan plateau, and to lay a foundation for the commercial production and application of this strain. The mycelial masses had herbicidal activity against the isolated leaves of the weeds Chenopodium album, Elsholtzia densa, Malva verticillata, and Amaranthus retroflexus L., with a descending herbicidal activity order of M. verticillata > E. densa > A. retroflexus > C. album. The fermentation broth also showed herbicidal activity by treating the whole plants of these four weeds in the descending order of C. album > E. densa > M. verticillata > A. retroflexus. Scanning electron microscopy revealed that GD-011-B mycelia invaded the inner part of the leaf of A. retroflexus through the stomata and epidermal tissue gap and extended to the back, damaging the leaf epidermis and tissues and sporulating on the leaf surface, ultimately resulting in leaf disease. The fermentation broth of pathogen GD-011-B was not pathogenic to Vicia faba L., Pisum sativum L., Hordeum vulgare L., or Triticum aestivum L., but it was slightly pathogenic to Brassica napus L. Therefore, Alternaria alternata can be used as a herbicidal microorganism with high potential for biological control of four major agricultural weeds on the Tibetan plateau.

Phytochemical Extracts of Dittrichia viscosa (L.) Greuter from Agroecological Systems: Seed Antigerminative Properties and Effectiveness in Counteracting Alternaria Leaf Spot Disease on Baby-Leaf Spinach.

Dittrichia viscosa (L.) Greuter subsp. viscosa (Asteraceae) is a perennial species naturally distributed in arid and marginal areas whose agroecological cultivation could be a useful innovation to produce quality biomass to extract phenolic-rich phytochemical blends. Here, biomass-yield trends were profiled at different growth stages under direct cropping, and inflorescences, leaves, and stems were submitted to water extraction and hydrodistillation. Then, four extracts were investigated for their biological activities in invitro and in planta assays. Extracts inhibited cress (Lepidium sativum)- and radish (Raphanus sativus)-seed germination and root elongation. All samples showed dose-dependent antifungal activity in the plate experiments, inhibiting up to 65% of the growth of the fungal pathogen Alternaria alternata, a leaf-spot disease agent of baby spinach (Spinacea oleracea). However, only the extracts from dried green parts and fresh inflorescences at the highest concentration significantly reduced (54%) the extent of Alternaria necrosis on baby spinach. UHPLC-HRMS/MS analysis revealed that the main specialized metabolites of the extracts are caffeoyl quinic acids, methoxylated flavonoids, sesquiterpene compounds such as tomentosin, and dicarboxylic acids, which may explain the observed bioactivity. Plant extracts obtained through sustainable methodology can be effective in biological agricultural applications.

Inhibitory effect of carvacrol against Alternaria alternata causing goji fruit rot by disrupting the integrity and composition of cell wall.

Goji (Lycium barbarum L.) is a widely planted crop in China that is easily infected by the pathogenic fungus Alternaria alternata, which causes rot after harvest. Previous studies showed that carvacrol (CVR) significantly inhibited the mycelial growth of A. alternata in vitro and reduced Alternaria rot in goji fruits in vivo. The present study aimed to explore the antifungal mechanism of CVR against A. alternata. Optical microscopy and calcofluor white (CFW) fluorescence observations showed that CVR affected the cell wall of A. alternata. CVR treatment affected the integrity of the cell wall and the content of substances in the cell wall as measured by alkaline phosphatase (AKP) activity, Fourier transform-infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). Chitin and β-1,3-glucan contents in cells decreased after CVR treatment, and the activities of β-glucan synthase and chitin synthase decreased. Transcriptome analysis revealed that CVR treatment affected cell wall-related genes in A. alternata, thereby affecting cell wall growth. Cell wall resistance also decreased with CVR treatment. Collectively, these results suggest that CVR may exert antifungal activity by interfering with cell wall construction, leading to impairment of cell wall permeability and integrity.

Rhizosphere bacteria associated with Chenopodium quinoa promote resistance to Alternaria alternata in tomato

Microorganisms can interact with plants to promote plant growth and act as biocontrol agents. Associations with plant growth-promoting rhizobacteria (PGPR) enhance agricultural productivity by improving plant nutrition and enhancing protection from pathogens. Microbial applications can be an ideal substitute for pesticides or fungicides, which can pollute the environment and reduce biological diversity. In this study, we isolated 68 bacterial strains from the root-adhering soil of quinoa (Chenopodium quinoa) seedlings. Bacterial strains exhibited several PGPR activities in vitro, including nutrient solubilization, production of lytic enzymes (cellulase, pectinase and amylase) and siderophore synthesis. These bacteria were further found to suppress the mycelial growth of the fungal pathogen Alternaria alternata. Nine bacterial strains were selected with substantial antagonistic activity and plant growth-promotion potential. These strains were identified based on their 16S rRNA gene sequences and selected for in planta experiments with tomato (Solanum lycopersicum) to estimate their growth-promotion and disease-suppression activity. Among the selected strains, B. licheniformis and B. pumilus most effectively promoted tomato plant growth, decreased disease severity caused by A. alternata infection by enhancing the activities of antioxidant defense enzymes and contributed to induced systemic resistance. This investigation provides evidence for the effectiveness and viability of PGPR application, particularly of B. licheniformis and B. pumilus in tomato, to promote plant growth and induce systemic resistance, making these bacteria promising candidates for biofertilizers and biocontrol agents.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6h) and a 1.46-fold upregulation during the infection hyphae formation period (8h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Antagonist Effect of Bacillus Species against Alternaria Rot of Tomato

The PGPR possesses antifungal activity against a broad range of pathogens. In this research study, the LPs of Bacillus strain B1 and B2 against Alternaria alternata were assessed. In vitro and in Planta assay on tomato fruit was made to check the post-harvest quality of fruits. The lipopeptides produced by both strains suppress the growth of the fungal pathogen Alternaria alternata and lessen the disease severity of the Alternaria rot on tomato fruits. The result revealed the quality of the Bacillus-treated fruits was also maintained and satisfactory. The lipopeptides of bacillus strain B1 and B2 represent a new innovative method to lessen the use of fungicides and chemical approaches to reduce the post-harvest loss caused by Alternaria alternata.

A zinc finger suppressor involved in stress resistance, cell wall integrity, conidiogenesis, and autophagy in the necrotrophic fungal pathogen Alternaria alternata

The tangerine pathotype of Alternaria alternata can withstand high-level reactive oxygen species (ROS). By analyzing loss- and gain-of-function mutants, this study demonstrated that a Cys2His2 zinc finger-containing transcription regulator, A. alternata Stress Response Regulator 1 (AaSRR1), plays a negative role in resistance to peroxides and singlet-oxygen-generating compounds. AaSRR1 plays no role in cellular susceptibility or resistance to superoxide-producing compounds. AaSRR1 also negatively regulates conidiogenesis, maintenance of cell wall and membrane integrities, and chitin biosynthesis. Some wild-type hyphae displayed necrosis after exposure to 30 mM H2O2, whereas AaSRR1 deficient mutant (ΔAaSRR1) hyphae had visible granules and vacuoles. sGFP-AaATG8 proteolysis assays revealed that H2O2 and starvation could trigger autophagy formation in both wild type and ΔAaSRR1. Autophagy occurred at higher rates in ΔAaSRR1 than wild type under both conditions, particularly after H2O2 treatments, indicating that autophagy might contribute to ROS resistance. Upon exposure to H2O2 or under starvation, AaSRR1 was translocated into the nucleus, even though the expression of AaSRR1 was decreased. AaSRR1 is required for vegetative growth but is dispensable for fungal virulence as assayed on detached calamondin leaves. AaSRR1 suppressed the expression of the gene encoding a HOG1 mitogen-activated protein (MAP) kinase implicated in ROS resistance. Mutation of AaSRR1 increased catalase activity but decreased superoxide dismutase activity, leading to fewer ROS accumulation in the cytosol. Nevertheless, our results indicated that AaSRR1 is a transcription suppressor for ROS resistance. This study also revealed tradeoffs between stress responses and hyphal growth in A. alternata.

Control of Early Blight Fungus (Alternaria alternata) in Tomato by Boric and Phenylboronic Acid.

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.

Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata.

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

The transcription regulator ACTR controls ACT-toxin biosynthesis and pathogenicity in the tangerine pathotype of Alternaria alternata

The host-selective ACT toxin is essential for the pathogenesis of the citrus fungal pathogen Alternaria alternata. However, the mechanism of ACT-toxin gene clusters ACT-toxin biosynthesis regulated by is still poorly understood. The biosynthesis of ACT toxin is mainly regulated by multiple ACT toxin genes located in the secondary metabolite gene cluster. In this study, we reported a transcription regulator ACTR contributes ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 in Alternaria alternata. We generated ACTR-disrupted and -silenced mutants in the tangerine pathotype of A. alternata. Phenotype analysis showed that the ACTR mutants displayed a significant loss of ACT toxin production and a decreased virulence on citrus leaves whereas the vegetative growth and sporulation were not affected, indicating an essential role of ACTR in both ACT toxin biosynthesis and pathogenicity. To elucidate the transcription network of ACTR, we performed RNA-Seq experiments on wild-type and ACTR null mutant and identified genes that were differentially expressed between two genotypes. Transcriptome profiling and RT-qPCR analysis demonstrated that the ACT toxin biosynthetic gene ACTS4 is down-regulated in ACTR mutant. We generated ACTS4 knock-down mutant and found that the pathogenicity of ACTS4 mutant was severely impaired. Interestingly, both ACTR and ACTS4 are not involved in the response to different abiotic stresses including oxidative stress, salt stress, cell-wall disrupting regents, and metal ion stress, indicating the function of these two genes is highly specific. In conclusion, our results highlight the important regulatory role of ACTR in ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 and underline the essential role of in the tangerine pathotype of A. alternata.

Genomic Sequence Resource of Alternaria alternata Strain B3 Causing Leaf Blight on Ophiopogon japonicus.

Black spot of Ophiopogon japonicus is an economically important disease, which can result in significant losses of both yield and quality of this traditional Chinese medicinal plant. The disease is caused by the small-spored fungal pathogen Alternaria alternata, a necrotrophic fungus that is ubiquitously distributed in the environment. Here, we present the draft whole-genome sequence of A. alternata strain B3 that caused black spot on O. japonicus. The assembly consists of 76 contigs with an estimated genome size of 33.8 Mb. Furthermore, we identified genes that may be associated with the pathogenicity, such as carbohydrate-active enzymes, secreted proteins, and secondary metabolite gene clusters. This genome resource will provide a useful source for future research on the evolution of pathogenicity of A. alternata and phylogenomic analysis in delineating phylogenetic lineages within the genus Alternaria.

Generation and characterization of atmospheric pressure dielectric barrier discharge air plasma and its antifungal potential: a case study on Alternaria alternata

The present research evaluates the generation and characterization of 26 kHz-atmospheric pressure dielectric barrier discharge (DBD) air plasma (DBD-AP) and the possible antifungal action against the pathogenic fungus Alternaria alternata. The DBD-AP visual and emission spectra intensities increased with increasing the applied voltage. The generated plasma was mainly inhomogeneous due to the presence of streamers in the discharge gap and it showed diffuse with less gap distance or with increasing the applied voltage. The measured consumed power was 26.52 W for 10.3 kV and 17.4 mA at 3 mm gap. The emission spectra showed the presence of NO and N2 bands which were enhanced due to the increase of the applied voltage. Significant reduction in A. alternata growth accompanied by loss of fungal cells structure was observed with increasing the DBD-AP exposure time. The present article confirms the potential of DBD-AP to efficiently control the plant-pathogenic fungus A. alternata.

Morphological and cultural studies of Alternaria alternata causing leaf spot and fruit rot of chilli

Leaf spot and fruit rot of chilli caused by Alternaria alternata (Fr.) Keissler is one of the most destructive disease of chilli (Capsicum annuum L.). Study was undertaken to conduct collection of samples, isolation, identification, pathogenicity, morphological and cultural characters of pathogen on different culture media in vitro. The isolations were made from the symptomatic samples collected from chilli plot from All India Coordinated Research Project on Vegetable Crops, Mahatma Phule Krishi Vidyapeeth, Rahuri. The isolation of fungal pathogen was done from affected samples of chilli leaf. Further purified and maintained on PDA media. Pathogenicity of test fungal pathogen was proved on healthy chilli plant in earthen pots under glasshouse conditions. The pathogen was identified as Alternaria alternata on the basis of morphological and cultural characteristics. The pathogen showed typical symptoms of leaf spot on chili in glasshouse. Alternaria alternata showed spots with concentric rings on leaves. Alternaria causes small reddish purple spots most characteristic symptom was brown to dark spots on older leaves. The circular spots enlarge and soon develop dark, concentric rings or ridges gaved target-like appearance. Lesion growth stopped by major leaf veins. Spotted leaves soon turn yellow, wither and died. In cultural studies, it was observed that the fungal test pathogen Alternaria alternata showed maximum radial growth and excellent sporulation on PDA media.

The basal transcription factor II H subunit Tfb5 is required for stress response and pathogenicity in the tangerine pathotype of Alternaria alternata.

The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss‐ and gain‐of‐function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA‐damaging agents, and oxidants. The expression of Aatfb5 was up‐regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall‐degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two‐hybrid and co‐immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase‐coding genes (AaCut2 and AaCut15) required for full virulence were down‐regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host.

Biotin biosynthesis affected by the NADPH oxidase and lipid metabolism is required for growth, sporulation and infectivity in the citrus fungal pathogen Alternaria alternata

The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of H2O2, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.