Nanodisks protect amphotericin B from ultraviolet light and oxidation‐induced damage

BACKGROUND: Macrolide polyene antibiotics possess potent broad‐spectrum antifungal properties. Use of these agents in the field or in controlled environments is impeded by their poor water solubility and susceptibility to oxidation‐ and/or light‐induced degradation. While typically used for human disease therapy, there is potential to expand the utility of polyene macrolide antibiotics, such as amphotericin B, for control of fungal disease infestation in agricultural settings. Thus, the susceptibility of this antibiotic to exposure‐induced activity loss was evaluated. RESULTS: Incubation of the prototype polyene amphotericin B (AMB) with phospholipid vesicles and apolipoprotein A‐I results in the formation of nanoscale complexes, termed nanodisks (NDs), capable of solubilizing significant quantities of AMB. To evaluate whether AMB incorporation into NDs conferred protection against light‐ or oxidation‐induced damage, yeast growth inhibition assays were conducted. Compared with AMB solubilized in detergent micelles, AMB incorporated into NDs was protected from damage caused by exposure to UV light as well as by KMnO₄‐induced oxidation. Furthermore, AMB‐NDs inhibited growth of the turfgrass fungus Marasmius oreades Fr. CONCLUSION: Results suggest that this water‐soluble formulation of a natural, biodegradable, antifungal agent represents a potential cost‐effective, non‐toxic and environmentally friendly substitute for chemical agents currently employed to control a range of fungal infestations. Copyright © 2009 Society of Chemical Industry     

The Effect of Nitrogen on Disease Development and Gene Expression in Bacterial and Fungal Plant Pathogens

Successful colonisation of plants by pathogens requires efficient utilisation of nutrient resources available in host tissues. Several bacterial and fungal genes are specifically induced during pathogenesis and under nitrogen-limiting conditions in vitro. This suggests that a nitrogen-limiting environment may be one of the cues for disease symptom development during growth of the pathogens in planta. Here we review recent literature on the effect of nitrogen and nitrogen-regulated genes on disease development, caused by phytopathogenic bacteria and fungi. Furthermore, the potential influence of nitrogen-limitation or general nutrient limitation on several in planta-induced bacterial and fungal pathogenicity, virulence and avirulence genes will be discussed.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Alternative hosts and plant tissues for the survival,sporulation and spread of the Ascochyta blight pathogen of chickpea

Various crop and weed species were infected naturally by Didymella rabiei (anamorph: Ascochyta rabiei) in blight-affected chickpea fields in the Palouse region of eastern Washington and northern Idaho, USA. The fungus was isolated from asymptomatic plants of 16 species commonly found in commercial crops in this region. Isolates of the pathogen from crop and weed species were pathogenic to chickpea and indistinguishable in cultural and morphological characteristics from isolates of D. rabiei from chickpea. Both mating types of D. rabiei were isolated from eight naturally infected plant species. Chickpeas were infected by D. rabiei when plants emerged through infested debris of seven crop and weed species. The teleomorph developed on overwintered tissues of seven plant species infected naturally by D. rabiei in a blight screening nursery and on debris of wheat, white sweet clover and pea inoculated with ascospores of D. rabiei or conidia of two compatible isolates of the pathogen. Didymella rabiei naturally infected 31 accessions of 12 Cicer spp. and the teleomorph developed on the overwintered debris of all accessions, including those of three highly resistant perennial species. The fungus developed on the stem and leaf pieces of ten plant species common to southern Spain inoculated with conidia of two compatible isolates of D. rabiei, and formed pseudothecia with asci and viable ascospores on six of ten species and pycnidia with conidia on all plant species.     

Pathogenicity of indole-3-acetic acid producing fungus <Emphasis Type="Italic">Fusarium delphinoides</Emphasis> strain GPK towards chickpea and pigeon pea

An indole-3-acetic acid (IAA) producing fungal strain was isolated from chickpea grown rhizospheric soil samples. Based on morphological and Internal Transcribed Spacer (ITS) region sequence analysis the new isolate was identified as Fusarium delphinoides. The Fusarium delphinoides strain produces and secretes IAA in-vitro as identified by HPLC and Mass spectrometry. The IAA production is dependent on tryptophan (Trp) as a nitrogen source in the medium. The IAA production is influenced by growth conditions such as pH of the medium, concentration of Trp and the nature of the carbon source. Additional nitrogen sources repress Trp dependent IAA production. Glucose and Trp served as the best carbon and nitrogen sources respectively. Pathogenicity of Fusarium delphinoides towards the plants was tested by electrolyte, nutrient leakage analysis and also by scoring the disease symptoms. Two cultivars of chickpea (ICCV-10 and L-550) and two cultivars of pigeon pea (Maruti and PT-221) were assessed for the pathogenicity by inoculating with spores of Fusarium delphinoides. The inoculation induced symptoms of Fusarium wilt as in the case of Fusarium oxysporum f. sp. ciceris (FOC), a known pathogen causing Fusarium wilt in chickpea. Electrolyte and nutrient leakage from the infected plants were used to assess the resistance, tolerance (moderately resistance) and susceptibility of the plants to the infection. Based on the results, both the pigeon pea cultivars (Maruti and PT-221) were rated as resistant, and ICCV-10 was rated as a tolerant cultivar of chickpea. However, chickpea cultivar L −550 was found to be a susceptible host for infection by Fusarium delphinoides. These results suggest that Fusarium delphinoides, which belongs to the Fusarium dimerum species group, is an IAA producing plant pathogen and causes wilt in chickpea. Further, along with pathogenicity tests, electrolyte and nutrient leakage analysis can be used to assess the pathogenicity of pathogenic fungi.     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

The mode of action of the antifungal agent phosphite1

Field studies have established that phosphorous acid (phosphite) is an effective control against a number of plant diseases. However, the mechanism of control is not known. We have used Phytophthora palmiuora to investigate the action of phosphorous acid as an antifungal agent. Phosphite is toxic to Phytophthoru in uitro, but concentrations which completely inhibit growth in vitro are rarely ever achieved in phosphite-treated plants. Phosphite concentrations (1 m mol 1^-1) which do not inhibit fungal growth in vitro cause changes in the metabolism and cell-wall components. We suggest that phosphite, when present in plants in concentrations too low to control fungi in vitro, may cause modification of the fungal cell surface in such a way that the plant is able to recognize it as foreign and respond by normal defence mechanisms.     

Fungitoxicity of 5-aminoisoxazole-4-thiocyanate derivatives

3-Methyl-5-(acyl/alkyl)aminoisoxazole-4-thiocyanates were prepared by rhodanation of N-acyl/alkyl-3-methyl-5-isoxazolamines. These products were tested for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. Some of the compounds showed interesting in-vitro activity. The more active compound in the in-vitro test displayed a moderate preventive activity against Plasmopara viticola, Alternaria solani and Pyricularia recondita.     

Functional diversity within the mitochondrial electron transport chain of plant pathogenic fungi

Mitochondrial transfer of electrons from NAD(P)H or FADH₂ to the terminal electron acceptor, oxygen, follows a highly complex scheme, involving numerous redox components. Whilst electron transfer has been extensively studied over past decades in mammalian, plant and some fungal species, relatively little is known about the respiratory chain of phytopathogenic fungi. The recent identification of the electron transport chain as a viable target for effective control of fungal infections has contributed to a significantly increased research effort into this area of fungal biochemistry. A striking feature of the electron transport chain within phytopathogenic fungi is the presence of components not found in the classical (mammalian) chain. Recent research has suggested the presence of a plant‐like ‘alternative’ oxidase, internal and external NAD(P)H dehydrogenases, and cyanide‐insensitive cytochrome c oxidases. In this mini‐review on electron transport in phytopathogenic fungi, the current status of research into the function and expression characteristics of these ‘alternative’ redox centres is discussed. © 2000 Society of Chemical Industry     

Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten

The phenolic composition of olive roots and stems was studied by high performance liquid chromatography–mass spectrometry. The in vivo levels of the principal phenolic compounds found in olive plants infected by Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten differed from the levels observed in non-infected plants. When the antifungal activity of these compounds against both fungi was studied in vitro, the most active were quercetin and luteolin aglycons, followed by rutin, oleuropein, p-coumaric acid, luteolin-7-glucoside, tyrosol and catechin. Microscopic study showed that these phenolic compounds affected the growth, morphology and ultrastructure of the fungi. Taken together, these findings suggest that the phenolic compounds present in olive plants play an active role in the protection against pathogen attack.     

Distribution of the Consensus Sequences of ABC Transporter Gene among Several Taxonomically Distinct Phytopathogenic Fungi

The distribution of ABC (ATP-binding cassette) transporter genes among several taxonomically distinct phytopathogenic fungi was investigated by Southern blot hybridization, polymerase chain reaction assays and partial sequencing. Consensus sequences of the ABC transporter gene, which might be concerned with multidrug resistance against fungicides and with pathogenicity in phytopathogenic fungi, were found in all of the examined phytopathogenic fungi, which belonged to the Mastigomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina. Received 24 August 2000/ Accepted in revised form 5 December 2000     

Hordenine is responsible for plant defense response through jasmonate-dependent defense pathway

Organic agriculture does not rely on synthetic chemical fungicides. An alternative pest management strategy to chemical fungicides is the use of bioactive natural compounds. Hordenine [4-(2-dimethylaminoethyl)] is a phenethylamine alkaloid found in barley. Although hordenine has various pharmacological effects, including antibiotic activity against microorganisms, no studies have been carried out to investigate the inhibitory effects of hordenine on phytopathogenic fungal infection in host plants. Both grape downy mildew and strawberry anthracnose were suppressed by hordenine treatment. Hordenine had no effect on mycelial growth of phytopathogenic fungi, whereas plant defense response through the jasmonate-dependent defense pathway was enhanced in hordenine-treated plants. The concern over environmental pollution has led to the introduction of new pesticides, including bioactive natural compound based pesticide. Hordenine may be used in organic agriculture as an innovative elicitor of plant defense response to downy mildew and anthracnose.     

取食不同寄主的草地贪夜蛾肠道微生物多样性分析

为绿色防控草地贪夜蛾Spodoptera frugiperda,基于16S rDNA和ITS高通量测序分析,对分别取食玉米、小麦以及田间常见杂草婆婆纳Veronica polita的草地贪夜蛾肠道细菌和真菌进行物种注释、多样性分析、物种组成分析及功能预测。结果显示,细菌注释共获得20门30纲72目106科164属214种,真菌注释共获得9门23纲46目90科154属237种。厚壁菌门、放线菌门和变形菌门为取食玉米、小麦和婆婆纳的草地贪夜蛾肠道优势细菌门,子囊菌门和担子菌门为取食玉米、小麦和婆婆纳的草地贪夜蛾肠道优势真菌门。肠球菌属Enterococcus为取食玉米、小麦和婆婆纳的草地贪夜蛾肠道细菌共同的优势属,相对丰度分别为70.86%、25.42%和72.02%;丝孢酵母属Tricho-sporon 和帚枝霉属 Sarocladium 为取食玉米的草地贪夜蛾肠道真菌的优势属,相对丰度分别为57.28%和17.38%,酵母菌目未分类属、小囊菌属Microascus和丝孢酵母属Trichosporon为取食小麦的草地贪夜蛾肠道真菌优势属,相对丰度分别为29.31%、27.01%和15.45%,双足囊菌科未分类属为取食婆婆纳的草地贪夜蛾肠道真菌优势属,相对丰度为61.79%。取食小麦的草地贪夜蛾肠道细菌群落的多样性最高,取食婆婆纳的草地贪夜蛾肠道真菌群落的多样性最高,取食婆婆纳的草地贪夜蛾肠道细菌和真菌群落的丰富度最高。取食玉米、小麦和婆婆纳的草地贪夜蛾肠道细菌群落功能主要与代谢通路相关,取食玉米、小麦和婆婆纳的草地贪夜蛾肠道真菌群落功能主要与基础代谢或生理功能相关。     

Arbuscular mycorrhizal fungal community structure and diversity are affected by host plant species and soil depth in the Mu Us Desert,northwest China

The vertical diversity and distribution of arbuscular mycorrhizal (AM) fungi were investigated in the Mu Us Desert, northwest China. Soils were sampled to 50?cm in depth in the rhizospheres of Hedysarum laeve, Artemisia ordosica, and Psammochloa villosa and 44 AM fungal species belonging to 10 genera were isolated. Several of these species have peculiar morphological features, which are distinct from other habitats. AM fungal diversity and distribution differed significantly among the three host plants and the five soil layers. Spore density, species richness, and the Shannon-Wiener index of AM fungi were 0.55–4.3 spores g^?1 soil, 7–36 and 1.78–2.89, respectively. Spore density and species richness had a significant positive correlation with soil total phosphorus content (0.0377–0.1129?mg?g^?1), and a negative correlation with soil pH (7.19–7.64). Nonmetric multidimensional scaling, PerMANOVA, and structural equation model analysis demonstrated that host plant species and soil depth significantly and directly influenced the structure of AM fungal communities. We concluded that diversity and distribution of AM fungi might be influenced by plant species, soil depth patterns, and soil nutrient availability in desert ecosystems. This research into AM fungal communities may lead to the development of AM fungi treatment for the mitigation of soil erosion and desertification using mycorrhizal plants, such as H. laeve, A. ordosica, and P. villosa.     

Role of host specificity in the speciation of <Emphasis Type="Italic">Ascochyta</Emphasis> pathogens of cool season food legumes

Ascochyta/legume interactions are attractive systems for addressing evolutionary questions about the role of host specificity in fungal speciation because many wild and cultivated cool season food legumes are infected by Ascochyta spp. and most of these fungi have described teleomorphs (Didymella spp.) that can be induced in the laboratory. Recent multilocus phylogenetic analyses of a worldwide sample of Ascochyta fungi causing ascochyta blights of chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris), and pea (Pisum sativum) have revealed that fungi causing disease on each host formed a monophyletic group. Host inoculations of these fungi demonstrated that they were host-specific, causing disease only on the host species from which they were isolated. In contrast to the strict association between monophyletic group and host observed for pathogens of cultivated legumes, Ascochyta fungi causing disease on wild bigflower vetch (Vicia grandiflora) were polyphyletic. Genetic crosses between several pairs of closely related, host-specific, and phylogenetically distinct Ascochyta fungi were fully sexually compatible. Progeny from these crosses had normal cultural morphology and segregation of molecular markers indicating a lack of intrinsic, post-zygotic mating barriers between the parental taxa. However, when progeny from a cross between a faba bean-adapted isolate (A. fabae) and a pea-adapted isolate (A. pisi) were assessed for their pathogenicity to the parental hosts, almost all progeny were non-pathogenic to either faba bean or pea. These results suggest that although these fungi have retained the ability to mate and produce progeny with normal saprophytic fitness, progeny are severely compromised in parasitic fitness. The host specificity of these fungi, coupled with the inability of hybrid progeny to colonize and reproduce on a host, may constitute strong extrinsic, pre-zygotic and post-zygotic mating barriers in these fungi and promote the genetic isolation and speciation of host-specific taxa. A phylogeny of the host plants is also being developed, and with more extensive sampling of pathogens and hosts from sympatric populations in the centre of origin, the hypothesis of cospeciation of pathogens and hosts will be tested. The objectives of this review are: (1) to summarize recent phylogenetic, host specificity and speciation studies of Ascochyta fungi, and (2) to suggest how current and future research using these pathosystems may lead to a better understanding of the role of host specificity in the speciation of plant-pathogenic fungi and the cospeciation of pathogens and their hosts.     

Total phenol,anthocyanin, and terpenoid content,photosynthetic rate,and nutrient uptake of Solanum nigrum L. and Digitaria sanguinalis L. as affected by arbuscular mycorrhizal fungi inoculation

Over the last decades, tillage, chemical fertilizers, and pesticides have reduced the beneficial fungal population size in arable soils. Though soil inoculation can be a practical way to restore arbuscular mycorrhizal fungi (AMF) population size, weeds may also be benefited, as well. This study was aimed to evaluate the effect of three AMF species (Funneliformis mosseae, Rhizoglomus fasciculatum, and Rhizoglomus intraradices) on photosynthetic rate, secondary metabolites content, reproductive organs percentage and nutrient uptake in Solanum nigrum L. and Digitaria sanguinalis L. weed species. Our results showed species variation in response to AMF inoculation, so that, while inoculation with R. intraradices fungal species decreased total biomass in S. nigrum plants significantly, it increased total biomass of D. sanguinalis plants by 26–49%. In addition, inoculation with F. mosseae species increased phenol, anthocyanin, and total terpenoid content in S. nigrum plants much more than D. sanguinalis. Increased photosynthetic rate, secondary metabolites content, and flowering percentage in AMF‐inoculated S. nigrum plants show the enhanced competitive ability and allelopathic potential of this weed when associated with AMF, which makes it a good competitor against other plant species in the environment.     

Identification of potential native chitinase-producing Trichoderma spp. and its efficacy against damping-off in onion

Chitinase-producing Trichoderma species have been recognized long ago against the phytopathogenic fungi. In this study, we evaluated the production of chitinase enzyme for seventeen isolates of Trichoderma isolated from onion growing districts of Punjab and assessed their bio-efficacy against damping-off in onion. In vitro, these Trichoderma isolates were screened for their antagonistic activity against the damping-off pathogen Fusarium oxysporum f.sp. cepae by dual culture assay. These isolates were also screened for their chitinase enzyme activity; it was found that isolates T5 and T8 are showing higher antagonistic activity on Fusarium oxysporum f.sp. cepae and also produced large amounts of chitinase enzymes in the presence of commercial colloidal chitin. The selected chitinolytic isolates were used in field studies to confirm the feasibility of their biological control efficacy against onion damping-off. In the field experiment, the seed+soil treatment of chitinolytic isolate (T8) showed a critical decrease of damping-off in onion by 88.75% over control.

     

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 Society of Chemical Industry     

The antifungal activity of polyoxin B and iprodione against phytopathogenic fungi recently isolated from diseased plants in Japan

A comparison was made of the minimum inhibitory concentrations (MICs) of polyoxin B and iprodione against Alternaria mali, A. kikuchiana and Botrytis spp. isolalated from diseased plants from various regions of Japan. Several of these fungal isolates differ markedly in their sensitivity to iprodione and polyoxin B. A study was made of the relationship between the MIC values of polyoxin B and iprodione against several isolates and disease control obtained when these materials were applied to plants infected with the same isolates.