Single Endophyte Research Articles

Endophytic Consortium With Diverse Gene-Regulating Capabilities of Benzylisoquinoline Alkaloids Biosynthetic Pathway Can Enhance Endogenous Morphine Biosynthesis in Papaver somniferum

Secondary metabolite biosynthesis in medicinal plants is multi-step cascade known to be modulated by associated endophytes. While a single endophyte is not able to upregulate all biosynthetic steps, limiting maximum yield achievement. Therefore to compliment the deficient characteristics in an endophyte we tried consortium of endophytes to achieve maximum yield. Here, efforts were made to maximize the in planta morphine yield, using consortium of two endophytes; SM1B (Acinetobacter sp.) upregulating most of the genes of morphine biosynthesis except T6ODM and CODM, and SM3B (Marmoricola sp.) upregulating T6ODM and CODM in alkaloid-less Papaver somniferum cv. Sujata. Consortium-inoculation significantly increased morphine and thebaine content, and also increased the photosynthetic efficiency of poppy plants resulted in increased biomass, capsule weight, and seed yields compared to single-inoculation. The increment in morphine content was due to the modulation of metabolic-flow of key intermediates including reticuline and thebaine, via upregulating pertinent biosynthetic genes and enhanced expression of COR, key gene for morphine biosynthesis. This is the first report demonstrating the endophytic-consortium complimenting the functional deficiency of one endophyte by another for upregulating multiple genes of a metabolic pathway similar to transgenics (overexpressing multiple genes) for obtaining enhanced yield of pharmaceutically important metabolites.

Genetic modification of asexual Epichlo\xeb endophytes with the perA gene for peramine biosynthesis

Development of grass–endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Interactions of arbuscular mycorrhizal and endophytic fungi improve seedling survival and growth in post-mining waste

The impact of fungal endophytes and the modulating role of arbuscular mycorrhizal fungi (AMF) on the vitality of Verbascum lychnitis, grown in the laboratory in a substratum from a post-mining waste dump was investigated. We report that inoculation with a single endophyte negatively affected the survival rate and biomass production of most of the plant-endophyte consortia examined. The introduction of arbuscular mycorrhiza fungi into this setup (dual inoculation) had a beneficial effect on both biomass yield and survivability. V. lychnitis co-inoculated with AMF and Cochliobolus sativus, Diaporthe sp., and Phoma exigua var. exigua yielded the highest biomass, exceeding the growth rate of both non-inoculated and AMF plants. AMF significantly improved the photosynthesis rates of the plant-endophyte consortia, which were negatively affected by inoculation with single endophytes. The abundance of PsbC, a photosystem II core protein previously shown to be upregulated in plants colonized by Epichloe typhina, exhibited a significant increase when the negative effect of the fungal endophyte was attenuated by AMF.

Identification of Volatile Metabolites from Fungal Endophytes with Biocontrol Potential towards <i>Fusarium oxysporum</i> F. sp. <i>cubense</i> Race 4

Problem statement: Fungal endophytes are widely studied for their potential as biocontrol agents towards fungal pathogens. In vitro assessments usually reveal their antibiosis and mycoparasitism nature, but little is understood regarding their production of volatile metabolites as mechanisms of antagonism. Approach: This study explored the potential of fungal endophytes in controlling the pathogen responsible for Fusarium wilt disease. Nine fungal endophytes were tested for their ability to inhibit the growth of the pathogenic Fusarium oxysporum F. sp. cubense race 4 (FocR4) via production of volatile inhibitory metabolites. The type of volatile metabolites produced were subsequently characterized and identified using the Gas-Chromatography Mass-Spectrophotometry (GCMS). Results: Eight of the isolates (BTF05, BTF07, BTF08, BTF15, BTF21, WAA03, WAA02, MIF01) showed positive results with percentages of inhibition varying from 1.43-31.43% while one isolate (ALF01), showed negative result (0% inhibition). Volatile profiles showed that these fungal endophytes produced between 15-47 volatile metabolites per isolate. However, the more volatile metabolites produced by a single endophyte does not indicate better biocontrol potential. Isolate BTF05 produced 47 different volatile metabolites, but has only 8.57% inhibition, compared to isolate BTF21 with 15 metabolites but a percentage of 11.43% inhibition. The potency of the volatile metabolites produced may also influenced the biocontrol potential of the fungal endophytes as some isolates such as BTF08 and MIF01 have only two to three known inhibitory metabolites but have higher PIDG values at 31.43 and 11.43%, respectively. Contrary, isolates WAA02 and WAA03 which has five to six metabolites but PIDG values of less than 3%. Conclusion: Fungal endophytes have the ability to produce several types of volatile metabolites to inhibit the growth of FocR4. These volatile inhibitory metabolites can be further extracted and the amount produced ascertained for future manipulation in biological control of FocR4.

Infection of tall fescue and perennial ryegrass plants by combinations of different Neotyphodium endophytes

Individual tall fescue (Festuca arundinacea) plants infected with both Neotyphodium coenophialum and N. lolii endophytes, and individual perennial ryegrass (Lolium perenne) plants infected with both N. lolii and Neotyphodium LpTG-2, were obtained following inoculation of naturally infected seedlings with the second endophyte. Differences in the ability of the endophytes to produce conidia, together with colony characteristics, enabled the endophytes in plants to be identified following incubation of excised leaf tissue on potato dextrose agar. Most tillers of dually infected plants were infected with just a single endophyte, but tillers infected with two endophytes were identified in three tall fescue plants. In these tillers one endophyte was always present at a much higher concentration than the other. Over time the incidence of dually infected tillers decreased and no tillers with both N. coenophialum and N. lolii were present 5 months after the associations were established. No evidence was obtained of exchange of nuclei between different endophytes present in dually infected tillers giving rise to heterokaryons or interspecific hybrids.

Distribution of fungal endophyte genotypes in doubly infected host grasses.

Fungal endophytes of the genus Epichloë live intercellulary in above ground plant parts of many pooid 'grasses of the temperate regions. The associations are characterized by single genotype entities since a given host individual normally contains a single endophyte genotype. They can persist over the life span of the hosts. This study examines whether two fungal genotypes can co-exist within a host plant, and how fungal genotypes are distributed within a host in the case of double infections. We selected four Epichloë bromicola strains that we identified as unique genotypes through RAPD' analysis. Young Bromus erectus plants, derived from callus cultures, were artificially inoculated with all possible double-strain mixtures of these fungal genotypes. For identification of fungal genotypes in planta, we designed genotype-specific primer pairs that flanked size-variable loci in the fungal genomes. Diagnostic PCR revealed that only one fungal genotype was present in most inoculated plants, but double infections were also observed with a frequency of 8% of all infected plants. Subsequent analyses of individual tillers of doubly infected plants revealed that, in a given tiller, both the leaf-blade and the leaf-sheath were colonized with only one endophyte genotype. Tillers without any detectable fungal DNA were also observed. Thus, co-existence of multiple endophyte genotypes within a single host plant is governed by mutual exclusion at the tiller level.

PLANT GROWTH RESPONSES TO VESICULAR‐ARBUSCULAR MYCORRHIZA

SUMMARYSeveral vesicular‐arbuscular mycorrhizal endophytes were inoculated separately on to onion plants in soils of low P status amended with five rates of Ca(H2PO4)2. H2O. Mycorrhizal infection was usually decreased by increasing additions of P, but the rate of root colonization and the infection plateau varied with different endophytes. Plant growth responses to mycorrhiza were large at low and medium P with Glomus mosseae and Glomus epigaeum and at medium P with Glomus macrocarpum and Gigaspora margarita. Glomus caledonium and Glomus sp.‘E3’were generally effective at low, medium and high P. Glomus clarum was ineffective at low P. The correlation between infection and growth response was higher for some endophytes than others. It was concluded that analysis of the VA symbiosis with any single endophyte cannot always be used as a basis for generalization about VA mycorrhizal fungi, and that the selection of field inoculants should take special account of endophyte behaviour in soils given extra phosphate.