Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.     

The genus Pythium in Taiwan, China (1) — a synoptic review

The genus Pythium,with slightly over 280 described species,has been classified traditionally with other filamentous,coenocytic,sporangia-producing fungi as "Phycomyetes".However,with recent advances in chemical,ultrastructural and molecular studies,Pythium spp.are now considered as "fungus-like organisms" or "pseudo-fungi" and are placed in the Kingdom Chromista or Kingdom Straminopila,distinct from the true fungi of the Kingdom Fungi or Kingdom Mycota.They are widely distributed throughout the world as soil saprophytes or plant pathogens.Because of the warm moist maritime climate,Taiwan,China,is especially rich in Pythium species.To date,48 species of Pythium have been reported from Taiwan,China,with the dominant species being Py.vexans,Py.spinosum,Py.splendens,Py.aphanidermatum,Py.dissotocum and Py.acanthicum.There is no definite geographical distribution of Pythium spp.in Taiwan,China.Twenty nine species of Pythium have proven to be plant pathogens attacking a wide variety of woody and herbaceous plants primarily causing pre-and post-emergence seedling damping-off,root rot,stem rot and rotting of fruits,tubers and ginger rhizomes,resulting in serious economic losses.The most important plant pathogenic species include Py.aphanidermatum and Py.Myriotylum,which are most active during the hot and wet summer months;whereas Py.splendens,Py.spinosum,Py.ultimum and Py.irregulare cause the greatest damage in the cool winter.Most Pythium spp.are non-specific pathogens,infecting mainly juvenile or succulent tissues.This review attempts to assess the taxonomic position of the genus Pythium and provide details of the historical development of the study of Pythium as pathogens in Taiwan,China,causing diseases of sugarcane,trees,vegetables,fruits,specialty crops and flowering plants,as well as measures to control these diseases.Of special note is the introduction of the S-H mixture which,when used as soil amendment,effectively controls many soil-borne Pythium diseases during the early stages of plant growth.The diversity of Pythium species in Taiwan,China,is discussed in comparison with the situation in the mainland of China and suggestions are made to fully utilize Pythium spp.as agents for biological control,in industry and medicine.     

Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation

In tomato soilless culture, slow filtration allows one to control the development of diseases caused by pathogenic microorganisms. During the disinfecting process, microbial elimination is ensured by mechanical and biological factors. In this study, system efficacy was enhanced further to a biological activation of filter by inoculating the pozzolana grains contained in the filtering unit with 5 selected bacteria. Three strains identified as Pseudomonas putida and 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years. These 5 bacteria displayed either a plant growth promoting activity (P. putida strains) or antagonistic properties (B. cereus strains). Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grains was particularly high as compared to the one observed in the control filter. Conversely to Bacillus spp. populations, Pseudomonas spp. ones remained abundant throughout the whole cultural season. The biological activation of filter unit very significantly enhanced fungal elimination with respect to the one displayed by the control filter. Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for the bacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month. Fast colonization of pozzolana grains by selected bacteria and their subsequent interaction with F. oxysporum are likely responsible for filter efficiency. Our results suggest that Pseudomonas spp. act by competition for nutrients, and Bacillus spp. by antibiosis and (or) direct parasitism. Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that of Fusarium since both filters demonstrated a high efficacy at the experiment start. Pythium spp. elimination appears to mainly rely on physical factors. It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteria failed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution. Their contribution to the establishment of a beneficial microbial community in the rhizosphere is discussed.     

Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.     

Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis

Cryptococcus neoformans var. neoformans is an important opportunistic fungal pathogen of patients whose immune system has been compromised due to viral infection, antineoplastic chemotherapy, or tissue transplantation. As many as 13% of all AIDS patients suffer a life-threatening cryptococcal infection at some time during the course of their HIV disease. To begin to understand the molecular basis for virulence in Cryptococcus neoformans var. neoformans serotype A, we have employed signature-tagged mutagenesis (STM) to identify mutants with altered virulence in a mouse model. The critical parameters of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed together in a single animal with each participating in the infection and that there is no apparent interaction between a virulent strain and an avirulent strain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecular analyses of these mutants indicated that 19 (49%) contained an insertion in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal insertion sites of those mutants that did not have an integration event in the actin promoter revealed an approximately random distribution among the chromosomes. Individual challenge of the putative mutants in a mouse model revealed five hypovirulent mutants and one hypervirulent mutant.     

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.     

The acquisition of hypovirulence in host-pathogen systems with three trophic levels

A major focus of research on the dynamics of host-pathogen interactions has been the evolution of pathogen virulence, which is defined as the loss in host fitness due to infection. It is usually assumed that changes in pathogen virulence are the result of selection to increase pathogen fitness. However, in some cases, pathogens have acquired hypovirulence by themselves becoming infected with hyperparasites. For example, the chestnut blight fungus Cryphonectria parasitica has become hypovirulent in some areas by acquiring a double-stranded RNA hyperparasite that debilitates the pathogen, thereby reducing its virulence to the host. In this article, we develop and analyze a mathematical model of the dynamics of host-pathogen interactions with three trophic levels. The system may be dominated by either uninfected (virulent) or hyperparasitized (hypovirulent) pathogens, or by a mixture of the two. Hypovirulence may allow some recovery of the host population, but it can also harm the host population if the hyperparasite moves the transmission rate of the pathogen closer to its evolutionarily stable strategy. In the latter case, the hyperparasite is effectively a mutualist of the pathogen. Selection among hyperparasites will often minimize the deleterious effects, or maximize the beneficial effects, of the hyperparasite on the pathogen. Increasing the frequency of multiple infections of the same host individual promotes the acquisition of hypovirulence by increasing the opportunity for horizontal transmission of the hyperparasite. This effect opposes the usual theoretical expectation that multiple infections promote the evolution of more virulent pathogens via selection for rapid growth within hosts.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Solid formulations of binucleate Rhizoctonia isolates suppress Rhizoctonia solani and Pythium ultimum in potting medium

Two isolates of binucleate Rhizoctonia spp., previously selected for efficacy in suppression of Rhizoctonia solani and Pythium spp., as well as plant growth promotion, were incorporated into various solid substrate formulations. These formulated products were assayed at three doses in three glass-house experiments for biocontrol of damping-off diseases in Capsicum annuum. R. solani anastomosis group 4 or Pythium ultimum var. sporangiiferum were incorporated into pasteurized potting medium with each formulated binucleate Rhizoctonia product. All formulations were effective against both pathogens in at least two experiments, but some formulations of one isolate of binucleate Rhizoctonia did not give consistent control of R. solani in one experiment. The most consistent formulation, which provided control of both pathogens at all doses of binucleate Rhizoctonia, was the simple substrate of rice hulls. The implications for commercialization of a biocontrol product are discussed.     

Evaluation of coumarin derivatives as anti-fungal agents against soil-borne fungal pathogens

Development of new and safer pesticides that are target-specific is backed by a strong Federal, public and commercial mandate. In order to generate a new generation of pesticides that are more ecologically friendly and safe, natural products are being evaluated for pesticidal activities. Many plant-derived chemicals have proven pesticidal properties, including compounds like sesamol (3,4-Methylenedioxyphenol), a lipid from sesame oil and coumarins (1,2-Benzopyrone) found in a variety of plants such as clover, sweet woodruff and grasses. Both of these plant-derived compounds have been shown to inhibit a range of fungi and bacteria and it is believed that these cyclic compounds behave as natural pesticidal defense molecules for plants. These compounds represent a starting point for the exploration of new derivative compounds possessing a range of antifungal activity and for use as seed protectants. Within this study, six derivatives of coumarin that resembled sesamol's structure were screened for their antifungal activity against a range of soil-bome plant pathogenic fungi. Fungi in this in vitro screen included Macrophomina phaseolina (causal agent of charcoal rot) and Pythium spp. (causal agent of seedling blight), two phylogenetically diverse and economically important plant pathogens. Preliminary studies indicate that many of these novel coumarin derivatives work very effectively in vitro to inhibit fungal growth and several coumarin derivatives have higher antifungal activity and stability as compared to either the original coumarin or sesamol compounds alone. Interestingly, several of these highly active coumarin derivatives are halogenated compounds with solubility in water, and they are relatively easy and inexpensive to synthesize. These halogenated coumarin derivatives remained active for extended periods of time displaying 100% inhibition of fungal growth for greater than 3 weeks in vitro. In addition to the in vitro fungal inhibition assays, preliminary phytotoxicity assays of these halogenated coumarin compounds show no obvious plant toxicity issues or interference in plant development. These results support additional research in this area of natural pesticide development.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR)

Biological control of soil-borne pathogens comprises the decrease of inoculum or of the disease producing activity of a pathogen through one or more mechanisms. Interest in biological control of soil-borne plant pathogens has increased considerably in the last few decades, because it may provide control of diseases that cannot or only partly be managed by other control strategies. Recent advances in microbial and molecular techniques have significantly contributed to new insights in underlying mechanisms by which introduced bacteria function. Colonization of plant roots is an essential step for both soil-borne pathogenic and beneficial rhizobacteria. Colonization patterns showed that rhizobacteria act as biocontrol agents or as growth-promoting bacteria form microcolonies or biofilms at preferred sites of root exudation. Such microcolonies are sites for bacteria to communicate with each other (quorum sensing) and to act in a coordinated manner. Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other Gram-negative bacteria. Several strains of the species Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. This progress will lead to a more efficient use of these strains which is worthwhile approach to explore in context of biocontrol strategies.     

Host Specificity of Pathogenic Pythium Species: Implications for Tree Species Diversity

In the Janzen–Connell hypothesis, host-specific natural enemies enhance species diversity and influence the structure of plant communities. This study tests the explicit assumption of host specificity for soil pathogens of the genus Pythium that cause damping-off disease of germinating seeds and seedlings. We isolated Pythium spp. from soil of a tropical forest in Panama. Then, in an inoculation experiment, we determined the pathogenicity of 75 tropical isolates of unknown pathogenicity and seven pathogenic temperate isolates of Pythium on seeds and/or seedlings of eight tropical tree species. Only three tropical isolates, one identified as P. ultimum and two as P. aphanidermatum , were pathogenic. Tropical pathogenic isolates were pathogenic on 4–6 of eight tree species. Temperate isolates were pathogenic on 0–4 of eight species, indicating that some tropical tree species are susceptible to novel isolates of Pythium . No tree species was susceptible to all isolates and two species were not susceptible to any isolate. Collectively, these results indicate that these Pythium isolates vary widely in their pathogenicity, causing differential mortality of potential host species; likewise, the tree species vary in their susceptibility to a given Pythium isolate. These differences in pathogenicity and susceptibility indicate some support for the Janzen–Connell assumption of host specificity. While they are not restricted to a single species, their intermediate level of specificity suggests that Pythium spp. have the potential to have some effect on forest community structure and diversity.     

Plant and fungal identity determines pathogen protection of plant roots by arbuscular mycorrhizas

1.  A major benefit of the mycorrhizal symbiosis is that it can protect plants from below-ground enemies, such as pathogens. Previous studies have indicated that plant identity (particularly plants that differ in root system architecture) or fungal identity (fungi from different families within the Glomeromycota) can determine the degree of protection from infection by pathogens. Here, we test the combined effects of plant and fungal identity to assess if there is a strong interaction between these two factors.
2.  We paired one of two plants ( Setaria glauca , a plant with a finely branched root system and Allium cepa , which has a simple root system) with one of six different fungal species from two families within the Glomeromycota. We assessed the degree to which plant identity, fungal identity and their interaction determined infection by Fusarium oxysporum , a common plant pathogen.
3.  Our results show that the interaction between plant and fungal identity can be an important determinant of root infection by the pathogen. Infection by Fusarium was less severe in Allium (simple root system) or when Setaria (complex root system) was associated with a fungus from the family Glomeraceae. We also detected significant plant growth responses to the treatments; the fine-rooted Setaria benefited more from associating with a member of the family Glomeraceae, while Allium benefited more from associating with a member of the family Gigasporaceae.
4.   Synthesis . This study supports previous claims that plants with complex root systems are more susceptible to infection by pathogens, and that the arbuscular mycorrhizal symbiosis can reduce infection in such plants – provided that the plant is colonized by a mycorrhizal fungus that can offer protection, such as the isolates of Glomus used here.     

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents.     

Fatty acid competition as a mechanism by which Enterobacter cloacae suppresses Pythium ultimum sporangium germination and damping-off

Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in beta-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.     

Ecology of Hymexazol-Insensitive Pythium Species in Field Soils

Soils from 100 irrigated fields (95 under vegetables, 5 under citrus) in different geographical locations in the West Bank (Palestinian Autonomous Territory) were surveyed for hymexazol-insensitive (HIS) Pythium species using the surface soil dilution plate (SSDP) method with the VP3 medium amended with 50 mg/L hymexazol (HMI) (VP3H50), over a period of 12 months. HIS Pythium species were isolated from 37% of the soils surveyed, with mean population levels ranging from 4.3-1422 CFU g(-1) dry weight. Eight HIS Pythium taxa were recovered on the VP3H50 medium, the most abundant of which was P. vexans (found in 29% of field soils surveyed). Seasonal variations in population levels of HIS Pythium species were studied in four fields over a period of 12 months. Significant seasonal variations in HIS population levels were detected in the four fields, with the highest population levels of HIS Pythium spp. encountered in spring and the lowest population levels in winter in three of the fields surveyed. Effects of HMI on linear growth and colony morphology of 149 Pythium ssp. isolates were examined on CMA amended with HMI at five concentrations. Pythium vexans isolates responded differently from those of the other Pythium species. Isolates of this important pathogen were more insensitive to HMI at high concentrations than the other main species tested. A large proportion of the P. ultimum isolates was either insensitive or weakly sensitive to HMI. Furthermore, a few isolates of other Pythium species were insensitive to the fungicide at various concentrations. The colony morphology of P. vexans isolates was not affected by HMI, whereas colonies of the other species showed sparse growth on the HMI amended medium relative to the control. The pathogenicity of P. vexans and P. ultimum isolates to cucumber seedlings was examined in growth chambers. Insensitive isolates of both species were found to be more virulent damping-off pathogens than the sensitive isolates. The present study demonstrates that HMI can not be used effectively in controlling Pythium spp. in soil inhabited with high densities of HIS Pythium spp. pathogens.     

The use of mutant and engineered microbial agents for biological control of plant diseases caused by Pythium: Achievements versus challenges

Pythium species are devasting pathogens causing major crop losses, e.g., damping-off in sugar beet caused by Pythium ultimum and root-rot of tomato caused by Pythium aphanidermatum. The use of natural antagonistic microorganisms is a promising environment-friendly approach to control Pythium-caused plant diseases. There are several examples of biocontrol of diseases caused by Pythium species but the application of bioeffectors (biological control agents) is limited for various reasons, including the restricted amount of gene-modification based biotechnological progress. The regulations in many countries prevent genetically modified bioeffectors from being routinely deployed in field conditions. Our two connected aims in this review are (1) to compile and assess achievements in genetic modification of bioeffectors which have been tested for parasitism or antagonism towards a Pythium plant pathogen or biocontrol of a plant disease caused by a Pythium species, and (2) discuss how a better performing bioeffector could be engineered to improve biocontrol of Pythium-caused plant diseases. We focus on the role of seven key mechanisms: cellulases, carbon catabolite de-repression, glycosylation, reactive oxygen species, chitin re-modelling, proteases, and toxic secondary metabolites. Genetic modifications of bioeffectors include gene deletion and overexpression, as well as the replacement of promoter elements to tune the gene expression to the presence of the pathogen. Gene-modifications are limited to fungal and bacterial bioeffectors due to the difficulty of gene modification in oomycete bioeffectors such as Pythium oligandrum. We assess how previous gene modifications could be combined and what other gene modification techniques could be introduced to make improved bioeffectors for Pythium-caused plant diseases. The broad host-range of Pythium spp. suggests engineering improved antagonistic traits of a bioeffector could be more effective than engineering plant-mediated traits i.e., engineer a bioeffector to antagonise a plant pathogen in common with multiple plant hosts rather than prime each unique plant host.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.