Host-specific Toxin Production Research Articles

Assessment of Pathogenicity in Helminthosporium maydis causing Southern Corn Leaf Blight Disease in the Region of Karnataka

Maize (Zea mays L) is the one of the most beneficial crops, adapted to various ecological and climatic states, it grades third after wheat and rice. Based on the research determinations for the last few years under the leadership of All India Coordinated Maize Improvement Project, 16 out of 61 diseases harmfully affecting this crop. One of the major diseases is Southern corn leaf blight (SCLB). The causative agent of the prevalent was recognized as the fungus Helminthosporium maydis. Research was carried out for pathogenicity assay. Pathogenicity assay was conducted with two methods, by collecting spores (2X105/ml), spraying on one month old maize plant. After 24 – 48 hours, it was found that spores collected from Davanagere (HMS3) and Kodagu (HMS5) region shows more yellow to brown lesion compare to all other regions. Second by extraction of toxin by methanol – chloroform method, purification by adsorption on charcoal and separated by using column chromatography and by thin layer chromatography. The Rf values, FTIR and UV absorption spectrum of purified toxin reveals the production of host specific toxin by H.maydis. Determination minimum toxic concentration required to satisfy the conditions as a host specific toxin. Keywords: Survey, Pathogenicity, Extraction, Host specific Toxin, Southern Corn Leaf Blight.

Coordinate control of oxidative stress tolerance, vegetative growth, and fungal pathogenicity via the AP1 pathway in the rough lemon pathotype of Alternaria alternata

Successful pathogenesis by the necrotrophic pathogen Alternaria alternata in citrus has been thought to be dependent on the production of host-specific toxins. This study determined whether the fungal pathogen can modulate reactive oxygen species (ROS) in the host plant by cloning and characterizing an RLAP1 gene in the rough lemon pathotype of A. alternata, which encodes a polypeptide that resembles many YAP1-like transcription activators of yeasts and fungi. Expression of the RLAP1 gene in a wild-type strain was responsive to many ROS-generating compounds. Mutants disrupted at the RLAP1 locus were hypersensitive to H 2O 2 and ROS-generating oxidants, showed reduced vegetative growth, were less effective in detoxifying H 2O 2, were non-pathogenic to rough lemon, and yet were unaltered in conidial formation or toxin production. When inoculated through wound sites, the mutants failed to incite any lesions. However, co-inoculation of the mutants with an NADPH oxidase inhibitor apocynin or diphenylene iodonium slightly restored fungal pathogenicity; introduction of the RLAP1 gene to a null mutant fully restored all phenotypes to wild type. Upon activation by H 2O 2, RLAP1::sGFP fusion protein is localized to the nucleus. Overall, the results reveal the essential role of effective detoxification via an RLAP1-mediated pathway in oxidative stress response and pathogenicity in A. alternata. It is proposed that the function of RLAP1 in A. alternata is to protect the pathogen from the toxicity of ROS generated by the citrus host.

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity.

An approach to the biosynthesis of the polyketide toxins excreted by the phytophagen fungus Botrytis cinerea

Botrytis cinerea is a well known pathogen of a number of commercial crops and produces many structurally diverse metabolites. There is no evidence for the production of host-specific toxins by this fungus, which is in accordance with the broad host range of this pathogen. Botrytis produces two series of phytotoxic metabolites: a family of characteristic sesquiterpene metabolites which contain the basic botryane skeleton, principally botrydial and dihydrobotrydial and two polyketide lactones types, botcinins and botrylactone [1,2,3]. The higher production of polyketide toxins from a botrydial sesquiterpene cyclase nock out mutant strain, led us to reinvestigate the structure and biosynthesis of these polyketide compounds.

Production of plants resistant to Alternaria carthami via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamustinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 105 spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min−1 mg−1 protein) from organogenesis and 47% (0.23 μmol min−1 mg−1 protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg−1 protein) and 19.5% (145 U mg−1 protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H2O2 generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H2O2 generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.

Automated and Unbiased Image Analyses as Tools in Phenotypic Classification of Small-SporedAlternariaspp.

ABSTRACT For more than 25 years, controversy has surrounded the characterization and differentiation of small-spored Alternaria spp. And, therefore, the application of names of several species that are involved in the pathology of diseases related to host-specific toxin production. The name A. alternata often has been broadly applied to various morphologically and chemically distinct groups of isolates from different hosts. The purpose of this study was to develop and evaluate automated and unbiased image analysis systems that will analyze different phenotypic characters and facilitate testing and application of the morphological species concept in Alternaria taxonomy. Host-specific toxin-producing Alternaria isolates assigned to five morpho-species were compared with representative isolates of morphologically distinct A. alternata. Combined results of growth rates at different temperatures, colony morphology, and metabolite profiles were found to be useful in characterization and differentiation of small-spored Alternaria spp. when standardized conditions are applied and representative isolates employed for comparison.

First Report of Leaf Spot on Japanese Plum Caused by an Alternaria sp. in Korea.

Japanese plum (Prunus salicina Lindley) is a deciduous tree in the family Rosaceae. In Korea, this plant is widely distributed in orchards as an important stone fruit as well as in gardens as an ornamental tree because of their abundant white blossoms. Every September to November since 2001, leaf spots were observed on Japanese plum in a garden in Cheongyang, Chungnam District, Korea. Early symptoms consisted of small, brown spots that were 2 to 5 mm in diameter. Later, the leaf lesions became circular or irregular, dark brown, expanded to 15 mm in diameter, and resulted in discoloration with necrosis on twisted leaves that was followed by defoliation. In November, older lesions sometimes appeared blackish brown as sporulation occurred on the lesions. The causal fungus was isolated from diseased leaves and cultured on potato dextrose agar. A culture has been placed in the CABI Herbarium (IMI Accession No. 387139). Conidial dimension averaged 34 × 12 μm. On the basis of morphological characteristics of conidia and conidiophores, the causal fungus was identified as a small-spored species of Alternaria as described by E. G. Simmons (1). Pathogenicity tests were conducted by inoculating slightly wounded and nonwounded leaves with a conidial suspension adjusted to 1 × 106 conidia/ml. Four leaves per each experiment were either wounded or not and inoculated with a spore suspension. The eight leaves were placed in a moist chamber at 25°C. After 6 to 10 days, small brown spots appeared on 87% of the wounded and nonwounded leaves. Control leaves sprayed with distilled water did not develop any symptoms. The causal fungus was consistently reisolated from the leaf spots. Results from pathogenicity tests were similar in a repeated test. It is possible that small-spored Alternaria spp. isolates are host specific (2). Eight Alternaria spp., including A. alternata, A. tenuis, A. tenuissima, and A. citri, have been found to cause black spot on fifteen Prunus spp. in China, Japan, Hong Kong, Libya, Mexico, Australia, and the United States (2). Further studies on the host-specific toxin production, geographical distribution, and host ranges for the species of Alternaria isolated from Japanese plum are in progress. To our knowledge, this is the first report of leaf spot on Japanese plum (P. salicina) caused by a small-spored Alternaria sp. in Korea.

Host-specific AB-toxin production by germinating spores of Alternaria brassicicola is induced by a host-derived oligosaccharide

The phytopathogenic fungus Alternaria brassicicola is the causal agent of black spot disease of Brassica plants and produces a host-specific protein toxin named AB-toxin. AB-toxin is released from spores germinating only on host leaves. In this study, we found a factor for inducing AB-toxin production that is released from host leaves after spore germination has begun. GC and GC–MS analyses of the compound purified by gel filtration chromatography demonstrated that it is an oligosaccharide of 1.3 kDa. This is the first evidence that host-specific toxin production by germinating spores of a fungal pathogen is induced by recognition of host-derived factors.

Toxin Production of Alternaria Alternata Tobacco Pathotype

ABSTRACTSome fungi of Alternaria spp. are known as foliar pathogens. Essential for this host range is the production of host-specific toxins, which are involved in the development in a few destructive diseases. A. alternata tobacco pathotype also produces host-specific and nonhost specific toxins. Toxic substances with host specific toxicity were released from germinating spores of this pathogen in 24 hr after germination. AT-toxin was partially purified from culture filtrate the fungus. It was shown to have host specific toxicity at cultivar level in leaf bioassay with tobacco cultivars Beinhart 1000.1, Shiroenshu 601 and Shiroenshu 1.

Tailor-made toxin target

The phytopathogenic fungus Alternaria alternata has a wide host range. However, certain pathotypes are selectively virulent and cause brown spots on leaves and fruits of specific species or cultivars of Citrus, whereas they are relatively harmless to others owing to the production of host-specific toxins. The so-called ACR toxin of the rough lemon pathotype (RLP) causes citrus brown spot disease exclusively on rough lemon. Physiological and ultrastructural data had previously suggested that mitochondria were the site of action of the ACR toxin. Kouhei Ohtani and colleagues [ 1. Ohtani K et al. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing. Proc. Natl. Acad. Sci. U. S.A. 2002; 99: 2439-2444 Crossref PubMed Scopus (52) Google Scholar ] have now provided a surprising explanation for the selectivity: the RNA of a mitochondrial gene (ACRS for ACR-toxin sensitivity) is specifically processed to yield a susceptible interaction with the ACR toxin.

Genetic Analysis of Pathogenicity and Host-specific Toxin Production of Alternaria alternata Tomato Pathotype by Protoplast Fusion

Several pathotypes of Alternaria alternata are known to produce host-specific toxins (HSTs) as agents of pathogenicity or virulence. However, investigations into the genetic controls of HST biosynthesis and pathogenicity of Alternaria pathogens have been limited by the lack of a sexual stage in the life cycle of these pathogens. We report here the development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST. Drug-resistant transformants have been isolated by genetic transformation of nonpathogenic A. alternata (strain O-94) and A. alternata tomato pathotype (strain As-27) with vectors conferring resistance to hygromycin B and geneticin, respectively. Protoplasts of the respective transformants were fused by polyethylene glycol treatment or electrofusion. Fusion products were selected by culturing in the presence of both hygromycin B and geneticin, then confirmed by amplification using a polymerase chain reaction with specific primers to the transforming drug-resistance genes. Stable fusants were purified by successive subcultures on selective medium and single-spore isolation. The resultant stable fusants, probably inter-strain hybrids, had the same pathogenicity and toxin production as the wild-type strain As-27. These results suggest that protoplast fusion has potential applications for genetic analysis of A. alternata pathogens.

Application of pulsed field gel electrophoresis to the study of genomes of fungal pathogens of plants

Pulsed field gel electrophoresis can determine karyotypes (chromosome size and number) of fungi that cannot be studied using cytological methods, due to their chromosomes failing to condense or being too small to visualise. This technique allows genome size and genetic maps to be developed for fungi that are not amenable to classical linkage analysis. Pulsed field gel electrophoresis has revealed that many fungal plant pathogens have highly variable genomes, even within individual species and that novel-sized chromosomes can be produced in some fungi during meiosis. It can also be exploited to demonstrate the presence of dispensable (B-type) chromosomes in fungi and it is a valuable tool in taxonomic studies for distinguishing between morphologically similar fungi. Pulsed field gel electrophoresis can be used to characterise complex loci, such as those controlling host-specific toxin production in Cochliobolus species. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed.

The structure of circinatin, a non-toxic metabolite from the plant pathogenic fungusPericonia circinata

A new non-toxic metabolite, circinatin, has been isolated from culture filtrates of the fungusPericonia circinata grown under modified conditions which suppress the normal production of host-specific toxins. The structure of the new compound has been established as in1 by combination of instrumental analysis and chemical degradation.

Relation between the Host-Specific Toxin Production and Pathogenicity in Alternaria alternata Pathogens

Relation between the Host-Specific Toxin Production and Pathogenicity in Alternaria alternata Pathogens

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogens. 3. Instability of pathogenicity in field isolates of A. alternata Japanese pear pathotype.

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogens. 3. Instability of pathogenicity in field isolates of A. alternata Japanese pear pathotype.

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogens. 4 Molecular cloning of mRNA in AK-toxin producing isolate.

Alternaria alternata Japanese pear pathotype produces a host-specific toxin (AK-toxin) as a determinant of host-specific pathogenicity. By employing a competitive hybridization procedure, specific transcripts were detected in a toxin producer, but not in its toxin-less avirulent mutant. Poly(A)+RNA fraction from the toxin producer was cloned as cDNA by Escherichia coli using pBR322 and screened by the competitive hybridization. The cDNA clones were obtained from transcripts which present in larger quantity in the producer than in its toxin-less mutant. Dot blot hybridization analysis by the cDNAs showed that there was a correlation between the amount of the specific RNAs and toxin productivity in several A. alternata isolates.

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogens. 2. Suppression of toxin production from germinating spores by chemical treatments.

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogens. 2. Suppression of toxin production from germinating spores by chemical treatments.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing alternaria black spot of strawberry. 1. Production of host-specific toxins and their biological activities.

The causal pathogen of Alternaria black spot of strawberry was identified as a distinct pathotype of Alternaria alternata (Fries) Keissler. Only one strawberry cultivar Morioka-16 was susceptible to the pathogen. Several cultivars of Japanese pear including Nijisseiki which were susceptible to A. alternata Japanese pear pathotype were also diseased by this pathogen. This fungus produced at least three species of host-specific toxins (named AF-toxin I, II and III, respectively). AF-toxin I and II were considered the major toxins. AF-toxin I induced necrosis and potassium leakage on the susceptible cvs. Morioka-16 and Nijisseiki. AF-toxin II induced only potassium leakage from cv. Morioka-16 but did not induce necrosis. AF-toxin II as well as AF-toxin I caused necrosis and potassium leakege from the cv. Nijisseiki.

Metabolic regulation of host-specific toxin production in Alternaria alternata pathogen. 1. Suppression of toxin production from germinating spores under high temperature stress.

The effect of thermal treatment on the pathogenicity of spores of Alternaria alternata Japanese pear pathotype was examined. When the virulent spores were subjected to a thermal treatment (e.g., at 50C for 10sec in water), they were not significantly inhibited their abilities of spore germination, elongation of germ tube and appressorium formation. However, they remarkably lost their pathogenicity to Japanese pear leaves susceptible to the black spot disease as well as productivity of host-specific AK-toxin during spore germination. Such an acquired effect on the pathogenicity and the toxin productivity was lost within about 24hr after the treatment. When the air-dried viable spores were continuosly exposed to hot summer temperatures for 13 days in field, the spores lost their pathogenicity and AK-toxin productivity without any visible damages on their germination ability. The above data suggest that certain thermal treatment of the spores of A. alternata Japanese pear pathotype causes to reduce their pathogenicity through the loss of AK-toxin productivity.