Fungal root endophytes of tomato from Kenya and their nematode biocontrol potential

The significance of fungal endophytes in African agriculture, particularly Kenya, has not been well investigated. Therefore, the objective of the present work was isolation, multi-gene phylogenetic characterization and biocontrol assessment of endophytic fungi harbored in tomato roots for nematode infection management. A survey was conducted in five different counties along the central and coastal regions of Kenya to determine the culturable endophytic mycobiota. A total of 76 fungal isolates were obtained and characterized into 40 operational taxonomic units based on the analysis of ITS, β-tubulin and tef1α gene sequence data. Among the fungal isolates recovered, the most prevalent species associated with tomato roots were members of the Fusarium oxysporum and F. solani species complexes. Of the three genes utilized for endophyte characterization, tef1α provided the best resolution. A combination of ITS, β-tubulin and tef1α resulted in a better resolution as compared to single gene analysis. Biotests demonstrated the ability of selected non-pathogenic fungal isolates to successfully reduce nematode penetration and subsequent galling as well as reproduction of the root-knot nematode Meloidogyne incognita. Most Trichoderma asperellum and F. oxysporum species complex isolates reduced root-knot nematode egg densities by 35–46 % as compared to the non-fungal control and other isolates. This study provides first insights into the culturable endophytic mycobiota of tomato roots in Kenya and the potential of some isolates for use against the root-knot nematode M. incognita. The data can serve as a framework for fingerprinting potential beneficial endophytic fungal isolates which are optimized for abiotic and biotic environments and are useful in biocontrol strategies against nematode pests in Kenyan tomato cultivars. This information would therefore provide an alternative or complementary crop protection component.     

Geoecology of Erratic Lichens of Xanthoparmelia vagans in an equatorial Andean Paramo

A population of 166 lichens of Xanthoparmelia vagans (Nylander) Hale, an obligatory erratic species, was investigated at 4530 m in the Venezuelan Andes. Lichen size and shape were strongly correlated; larger specimens were less spherical and compact than smaller ones. This was related to a greater frequency of disturbance – mainly by wind and frost – of small lichens, which are transported more easily than larger, heavy ones. Constant movement produces nearly spherical specimens, but as lichens grow larger, they are rotated less frequently and rest on a single side for increasingly longer periods; these immobile lichens become progressively more flattened and lenticular in shape. Many large lichens had two distinctive sides, an upper and a lower one, with different coloration. The upper face was pale yellowish green to olive gray (Munsell 5Y 8/4–5/2), the lower one black to very dark brown (10YR 2/1–2/2). Small lichens usually showed no differences between sides. Color variation is also induced by lichen movement. In frequently rotating lichens, thalli grow evenly in all directions due to a uniform insolation. In stationary lichens, thalli remain permanently oriented facing upwards, and their upper and lower sides diverge in coloration. The largest lichens break up easily. Some large specimens were in the process of fragmentation; these had a distinctive kidney-shaped form. Experimental splitting of 16 lichens showed that reduction in size by fragmentation also results in more equant specimens than the original ones. Laboratory experiments on aeolian transport indicated that wind speeds in the paramo are capable of carrying the erratic lichens, but soil areas covered by soil nubbins (miniature mounds produced by needle ice) can trap moving lichens, and greater wind velocities are then needed to re-mobilize them. Lichens were able to quickly absorb and store large amounts of water. Large lichens retained less water than small ones, but lost moisture at much slower rates. Needle-ice growth delivers water to the paramo soil surface, where lichens can readily imbibe it. The water relations of Xanthoparmelia, considered to be ecologically crucial for lichen growth and survival, are discussed in detail.     

Use of endophytes as biocontrol agents

Plant diseases, caused by various microorganisms, including viruses, bacteria, fungi, protozoa and nematodes, affect agricultural practices and result in significant crop losses. Fungal pathogens are the major cause of plant diseases and infect most plants. Agrochemicals play a significant role in plant disease management to ensure a sustainable and productive agricultural system. However, the intensive use of chemicals has adverse effects on humans and ecosystem functioning and also reduces agricultural sustainability. A sustainable agriculture is achieved through reduction or elimination of fertilizers and agrochemicals, resulting in minimal impact to the environment. Recently, the use of antagonistic endophytes as biocontrol agents is drawing special attention as an attractive option for management of some plant diseases, resulting in minimal impact to the environment. Endophytes that resides asymptomatically within a plant, have the potential to provide a source of candidate strains for potential biocontrol applications. This review addresses biocontrol methods using endophytic fungi such as Colletotrichum, Cladosporium, Fusarium, Pestalotiopsis and Trichoderma species as an attractive option for management of some plant diseases. Potential endophytes are screened in vitro and in vivo to test their antagonistic actions by different mechanisms, including mycoparasitism, production of lytic enzymes and/or antibiotics and induction of plant defenses. Currently, efforts are being made to commercialize these biocontrol agents. A continued research pipeline consisting of screening, in vitro and in vivo testing, biomass production and commercialization of endophytes as biocontrol agents may contribute to sustainable agriculture.     

Taxol from fungal endophytes and the issue of biodiversity

Fungi represent one of the most understudied and diverse group of organisms. Commonly, these organisms make associations with higher life forms and may proceed to biochemically mimic the host organism. An excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in the plant genusTaxus (yew). However, taxol has been reported in a novel endophytic fungus—Taxomyces andreanae, but also has been demonstrated to occur in a number of unrelated fungal endophytes includingPestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia and others. Thus, this report presents information on the presence of taxol among disparate fungal genera, and uses these observations as an additional argument to support efforts to study fungal endophytes and preserve their associated host plants.     

Biocontrol potential of endophytes harbored in Radula marginata (liverwort) from the New Zealand ecosystem

Radula marginata and Cannabis sativa L. are two phylogenetically unrelated plant species containing structurally similar secondary metabolites like cannabinoids. The major objective of our work was the isolation, identification, biocontrol efficacies, biofilm forming potential and anti-biofilm ability of endophytic microbial community of the liverwort R. marginata, as compared to bacterial endophytic isolates harbored in C. sativa plants. A total of 15 endophytic fungal and 4 endophytic bacterial isolates were identified, including the presence of a bacterial endosymbiont within an endophytic fungal isolate. The endosymbiont was visible only when the fungus containing it was challenged with two phytopathogens Botrytis cinerea and Trichothecium roseum, highlighting a tripartite microbe–microbe interaction and biocontrol potency of endophytes under biotic stress. We also observed sixteen types of endophytic fungal-pathogen and twelve types of endophytic bacterial-pathogen interactions coupled to varying degree of growth inhibitions of either the pathogen or endophyte or both. This showed the magnitude of biocontrol efficacies of endophytes in aiding plant fitness benefits under different media (environmental) conditions. Additionally, it was ecologically noteworthy to find the presence of similar endophytic bacterial genera in both Radula and Cannabis plants, which exhibited similar functional traits like biofilm formation and general anti-biofilm activities. Thus far, our work underlines the biocontrol potency and defensive functional traits (in terms of antagonism and biofilm formation) of endophytes harbored in liverwort R. marginata as compared to the endophytic community of phylogenetically unrelated but phytochemically similar plant C. sativa.     

Antifungal metabolites from fungal endophytes of Pinus strobus

The extracts of five foliar fungal endophytes isolated from Pinus strobus (eastern white pine) that showed antifungal activity in disc diffusion assays were selected for further study. From these strains, the aliphatic polyketide compound 1 and three related sesquiterpenes 2-4 were isolated and characterized. Compound 2 is reported for the first time as a natural product and the E/Z conformational isomers 3 and 4 were hitherto unknown. Additionally, the three known macrolides; pyrenophorol (5), dihydropyrenophorin (6), and pyrenophorin (7) were isolated and identified. Their structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data where available. The isolated compounds 1, 2, and 5 were antifungal against both the rust Microbotryum violaceum and Saccharomyces cerevisae.     

Systematic screening strategy for fungal laccase activity of endophytes from Otoba gracilipes with bioremediation potential

Fungal laccases are promising for biotechnological applications, including bioremediation and dye biotransformation, due to their high redox potential and broad substrate specificity. However, current bioprospecting methods for identifying laccase-producing fungi can be challenging and time-consuming. For early detection, it was developed a three-step, multi-criteria weighting system that evaluates fungal strains based on: First, the biotransformation capacity of three dyes (i.e., Congo red, brilliant blue G-250, and malachite green), at three different pH values, and with a relative weighting supported for the redox potential of each colorant. The relative decolorization coefficient (RDC), used as th2e first classification criterion, expressed their potential performance. Second, under the same conditions, laccase activity was estimated by observing the different degrees of oxidation of a given substrate. The selection criterion was the relative oxidation coefficient (ROC). Finally, laccase activity was quantified in submerged fermentations using three inducers (i.e., loofah sponge, Tween 80, and veratyl alcohol). This multicriteria screening strategy evaluated sixteen isolated endophytic fungal strains from Otoba gracilipes. The system identified Beltraniopsis sp. ET-17 (at pH values of 5.00 and 5.50) as a promising strain for dye biotransformation, and Phlebia floridensis as the best laccase producer, achieving a high activity of 116 μmol min⁻¹ L⁻¹ with loofah sponge as an inducer. In-vitro testing confirmed the efficacy of P. floridensis, with 53.61 % decolorization of a dye mixture (brilliant blue-Congo red. ratio 1:1) after 15 days of incubation. Thus, with the proposed screening strategy it was possible to highlight two species of interest at an early bioprospecting stage on a Colombian native tree poorly explored.     

Rhizobia: a potential biocontrol agent for soilborne fungal pathogens

Rhizobia are a group of organisms that are well known for their ability to colonize root surfaces and form symbiotic associations with legume plants. They not only play a major role in biological nitrogen fixation but also improve plant growth and reduce disease incidence in various crops. Rhizobia are known to control the growth of many soilborne plant pathogenic fungi belonging to different genera like Fusarium, Rhizoctonia, Sclerotium, and Macrophomina. Antagonistic activity of rhizobia is mainly attributed to production of antibiotics, hydrocyanic acid (HCN), mycolytic enzymes, and siderophore under iron limiting conditions. Rhizobia are also reported to induce systemic resistance and enhance expression of plant defense-related genes, which effectively immunize the plants against pathogens. Seed bacterization with appropriate rhizobial strain leads to elicitation and accumulation of phenolic compounds, isoflavonoid phytoalexins, and activation of enzymes like L-phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), peroxidase (POX), polyphenol oxidase (PPO), and others involved in phenylpropanoid and isoflavonoid pathways. Development of Rhizobium inoculants with dual attributes of nitrogen fixation and antagonism against phytopathogens can contribute to increased plant growth and productivity. This compilation aims to bring together the available information on the biocontrol facet of rhizobia and identify research gaps and effective strategies for future research in this area.     

Non-systemic fungal endophytes of grasses

Many fungi behave as endophytes in grasses. Unlike the well known Epichloë/Neotyphodium species, most other endophytes are not capable of systemic colonization of plant organs, or seed transmission. The species diversity of the non-systemic endophytic mycobiota of grasses is large, dominated by ascomycetes. The relative abundance of species is very unequal, a few dominant taxa like Acremonium, Alternaria, Cladosporium, Epicoccum and Penicillium spp., occur in many grasses and locations. In contrast, many rare species are isolated only once in endophyte surveys. The possible ecological functions of endophytes are diverse, and often unknown. Latent pathogens represent a small fraction of endophytic mycobiotas, indicating that many non-pathogenic fungal taxa are able to enter plants overriding defence reactions. Some dominant species behave as latent saprotrophs, sporulating when the host tissue dies. Endofungal viruses and bacteria occur among endophytic species, but their effect in their hosts is largely unknown.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

Current perspectives on the volatile-producing fungal endophytes

Microbial-derived volatiles are ubiquitous in the environment and actively engaged in bio-communication with other organisms. Recently, some volatile-producing endophytes (VPEs), cryptic fungal symbionts persisting in healthy plant tissues, have attracted great attention due to their strong antibiotic activity or production of carbon chains that are identical to many of those found in petroleum, while other fragrant volatiles can be used in the flavoring industries. From an application-oriented and biotechnological point of view, these findings show significant promise for sustainable development of agriculture, forestry, and industry, especially in the control of fruit postharvest diseases, soil-borne pathogen management, and bio-fuel production. In comparison, the ecological importance of VPEs has only rarely been addressed and warrants further exploration. In this review, we summarize the current knowledge and future directions in this fascinating research field, and also highlight the constraints and progresses towards commercialization of VPEs products.     

Physiological and molecular characterization of Phytophthora infestans isolates from the Central Colombian Andean Region

BackgroundLate blight, caused by Phytophthora infestans, is one of the most devastating diseases found in potato and tomato crops worldwide. In Colombia it also attacks other important crops: cape gooseberry and tree tomato. The knowledge of the pathogen population is determinant to effectively design control strategies.AimsTo determine the physiological and molecular characteristics of a set of Colombian P. infestans isolates.MethodsStrains isolated from Cundinamarca and Boyacá were examined for the level of resistance to mefenoxam and cymoxanil. Virulence was tested for all strains and crosses between A1 mating type, from different hosts, and the Colombian A2 mating type were tested for the production and viability of oospores in different substrates. Additionally, the molecular diversity of the avirulence gene Avr3a, the β-tubulin gene, and two single copy genes showing RxLR motif, was assessed.ResultsWe found all levels of mefenoxam sensitivity, with 48% of the strains resistant. A high diversity of races was detected and the population was genetically clonal. Colombian strains had the possibility of sexual reproduction.ConclusionsThese results will help in optimizing the use of fungicides and deployment of resistance as control strategies and will contribute to broader studies on diversity of this pathogen.     

Effect of potential biocontrol agents selected among grapevine endophytes and commercial products on crown gall disease

The current strategies for the control of Agrobacterium vitis crown gall in grape are generally unsuccessful once the pathogen has established in vineyards. Experimental trials were conducted to evaluate the effectiveness of treatments based on non-pathogenic endophytes isolated from asymptomatic grapevines growing in vineyards with high incidence of crown gall and on microorganisms isolated from commercial products. Two-year in planta trials conducted on rootstocks treated with endophytic isolates showed the effectiveness of two bacterial endophytes, both in the genus Curtobacterium, and one fungal isolate in the genus Acremonium in reducing crown gall development. For the commercial biological control agents, Bacillus subtilis SR63 and Trichoderma asperellum T1 were the most effective strains against A. vitis, indicating commercial products could be reserves to draw upon to identify useful biocontrol agents. Based on the combination of data in this work, microorganisms, both endophytes and those formulated in commercial products, were identified that can potentially be exploited for the control of grapevine crown gall disease.     

禾草内生真菌作为生防因子的潜力分析

早熟禾亚科多种禾草可与Neotyphodium内生真菌形成禾草-内生真菌共生体, 这种植物-微生物共生体性状较为稳定, 且在自然界中广泛存在。禾草-内生真菌共生体稳定的互利共生关系不但保证了内生真菌所需的全部营养物质, 而且共生体产生的次生代谢物又可显著提高宿主禾草对生物胁迫的抗逆性。众多研究表明, 内生真菌的侵染可显著提高宿主禾草对虫害、病害及伴生植物等多种生物胁迫的抗性。据不完全统计, 禾草内生真菌对蛛形纲、线虫纲、昆虫纲3个纲至少79个种的害虫表现出较明显的抗性, 对至少22个种的病原真菌表现出明显的抗性。尽管利用内生真菌进行禾草品种选育及其品质改良的技术日趋成熟, 但是内生真菌在不同宿主禾草之间高效的替代转化技术, 及其在宿主体内遗传的稳定性仍有待于进一步深入探索。研究者把禾草内生真菌作为生防手段, 在未来的应用过程中不应只考虑其与宿主禾草之间的共生特异性, 而应更全面地分析禾草-内生真菌-生态环境之间的相互关系, 让内生真菌更好地为人类服务。     

喜树内生菌与喜树碱的关系

通过对喜树幼苗中喜树碱含量的分析,发现不同生长期、不同器官中喜树碱的含量不同,幼叶和根中喜树碱的含量较高。虽然喜树中含有对真核细胞具有毒性作用的喜树碱,但仍有12种内生菌从喜树的不同器官中分离出来。内生菌对喜树碱的敏感性实验表明,10μg／mL喜树碱对2种内生菌的生长几乎没有抑制作用,即是100μg／mL浓度的喜树碱对它们的生长抑制也是有限的。     

Potential analysis of grass endophytes Neotyphodium as biocontrol agents

Many grasses in the subfamily Pooideae develop symbioses with Neotyphodium fungal endophytes, which exist widely in nature. The stably symbiotic relationship not only ensures accessible nutrients required by Neotyphodium fungal endophytes, but also significantly increases the resistance of host grasses to biological stresses through the production of secondary metabolites. Previous studies show that infected grasses with endophytic fungi have prominently enhanced resistance to pests, plant diseases, companion plants and other biological stresses. Grass endophytic fungi show remarkable resistant to at least 79 species of pests from three classes; arachnida, nematode and insecta, and to at least 22 species of pathogenic fungi. Although the biotechnological application of endophytic fungi in grass breeding for variety selection and quality improvement has progressed well, opportunities remain for further exploring the use of fungal endophytes among different host grasses coupled with the examination of genetic stability of Neotyphodium in novel host grasses. In the future application of endophytic fungi as a bio-control method, researchers should not only consider specificities of host grasses, but also need to have comprehensive analysis and knowledge about the mutual relationships among grasses, endophytic fungi and ecological environments, which will help use endophytic fungi to better serve humanity.     

A fungal pathogen with potential for biocontrol ofStriga hermonthica (Scrophulariaceae)

Fusarium equiseti (Corda) Saccardo (IMI Number 311922b) was isolated from diseasedStriga hermonthica (Del.) Bentham parasitic on sorghum in the Nuba Mountains Rural Development Project, Kordofan, Sudan in November 1986. Symptoms of the disease consisted of progressive blackening and drying of the leaves and flowers. The disease caused 50% mortality ofS. hermonthica populations in two areas.     

Characterization of cellulases of fungal endophytes isolated from Espeletia spp.

Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production.