Iñigo Zabalgogeazcoa

Endophytic mycobiota of leaves and roots of the grass Holcus lanatus

Holcus lanatus is a grass that grows in humid, often waterlogged soils in temperate zones around the world. The purpose of this work was to identify fungal endophytes associated with its roots and leaves, and to describe the diversity and spatial distribution patterns found in its mycobiota. Holcus plants were sampled at 11 locations in western and northern Spain, and endophytes were isolated from leaves and roots of each plant. Morphological and molecular methods based on the ITS1-5.8SrRNA-ITS2 sequence were used for isolate identification. In total, 134 different species were identified, 77 occurred in leaves, 79 in roots, and 22 were common to both organs. The dominant species of the mycobiota were isolated from roots and leaves, and were species generally considered as multi-host endophytes. The species richness was similar in leaves and roots, but the composition of isolates from roots varied more among locations than in leaf mycobiotas, suggesting that soil characteristics may have strongly influenced the root mycobiota. Significant variations with respect to the composition of their mycobiota among different locations indicate that beta diversity is a first order factor governing the richness and distribution of the endophytic mycobiota in grasses.

Genetic structure of natural populations of the grass endophyte Epichloë festucae in semiarid grasslands

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (FST: 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.

Mycoviruses are common among different species of endophytic fungi of grasses.

A survey of mycoviruses was made in a collection of 103 isolates belonging to 53 different species of endophytic fungi of grasses. Double-stranded RNA (dsRNA) elements were detected in isolates of 12 of the species analyzed. The banding characteristics and sizes of some of the dsRNA elements suggest that they might belong to previously described mycovirus families. The observed incidence (22.6%) indicates that the presence of mycoviruses could be common among species of this group of ubiquitous fungi.

Mycoviruses infecting the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum.

A mixed virus infection in a strain of the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum was deduced from a study of the transmission to conidia of several double-stranded RNA (dsRNA) elements. The transmission rates of each dsRNA were different, and monosporic isolates harbouring different combinations of the original set of six dsRNAs were obtained. A 5196 bp dsRNA element was sequenced and represents the genome of T. cylindrosporum virus 1 (TcV1), a new member of the genus Victorivirus in the Totiviridae family. This virus was transmitted to 81.4% of the conidia; in contrast, four dsRNAs of 3.1-3.7 kbp were transmitted only to 4.7% of the monosporic isolates obtained from the infected parental strain. These four dsRNAs did not show segregation during transmission, and one of them was shown by sequence analysis to encode an RdRp, suggesting that the four molecules might represent the whole genome of a quadripartite chrysovirus. A third possible virus with a genome of approximately 4.2 kbp was transmitted to 79.1% of the monosporic isolates produced by the infected strain. Ribavirin was used to cure T. cylindrosporum from viruses, and TcV1 was sensitive to this drug. All monosporic cultures derived from the infected strain treated with 80 and 100 μM concentrations of the drug were free of TcV1.

Prevalence and Diversity of Viruses in the Entomopathogenic Fungus Beauveria bassiana

ABSTRACT Viruses have been discovered in numerous fungal species, but unlike most known animal or plant viruses, they are rarely associated with deleterious effects on their hosts. The knowledge about viruses among entomopathogenic fungi is very limited, although their existence is suspected because of the presence of virus-like double-stranded RNA (dsRNA) in isolates of several species. Beauveria bassiana is one of the most-studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as a biological control agent against invertebrates in agriculture. We analyzed a collection of 73 isolates obtained at different locations and from different habitats in Spain and Portugal, searching for dsRNA elements indicative of viral infections. The results revealed that the prevalence of viral infections is high; 54.8% of the isolates contained dsRNA elements with viral characteristics. The dsRNA electropherotypes of infected isolates indicated that virus diversity was high in the collection analyzed and that mixed virus infections occurred in fungal isolates. However, a hybridization experiment indicated that dsRNA bands that are similar in size do not always have similar sequences. Particular virus species or dsRNA profiles were not associated with locations or types of habitats, probably because of the ubiquity and efficient dispersion of this fungus as an airborne species. The sequence of one of the most common dsRNA elements corresponded to the 5.2-kbp genome of a previously undescribed member of the Totiviridae family, termed B. bassiana RNA virus 1 (BbRV1).

An Epichloë endophyte affects the competitive ability of Festuca rubra against other grassland species

Background and aimsFestuca rubra plants are asymptomatically infected by the systemic fungal endophyte Epichloë festucae and high infection rates have been found in natural grasslands. Our objective was to determine the effect of endophyte infection on the competitive ability of Festuca rubra in binary mixtures against other grassland species.MethodsIn two glasshouse experiments, endophyte-infected (E+) and non-infected (E−) plants of two half-sib lines (PEN, RAB) of Festuca rubra were grown in monocultures and in binary mixtures against five grassland species: Trifolium repens, Trifolium pratense, Trifolium subterraneum, Lotus corniculatus and Plantago lanceolata.ResultsBiomass production of F.rubra was affected by endophyte infection and plant line but not by competition with T.pratense. In the PEN line E+ plants produced less root biomass than E−; and in the RAB line E+ plants produced less shoot biomass than E−. In spite of these differences shoot and root biomass production of Trifolium pratense were more inhibited when growing with endophyte-infected plants of Festuca rubra than with E− plants, regardless of the plant line. In both F.rubra lines, the endophyte increased the phosphorus concentration in shoots. Regardless of F.rubra plant line, root biomass of the five target species was more inhibited when growing with endophyte-infected than with endophyte-free plants.ConclusionsIrrespective of F.rubra line, endophyte-infected plants had a better competitive ability than non-infected plants, as indicated by the lower relative yield of companion plants when growing in mixture with E+ plants. We suggest an allelopathic effect of E+ red fescue on other grassland species.

Fungal Endophyte (Epichloë festucae) Alters the Nutrient Content of Festuca rubra Regardless of Water Availability

Festuca rubra plants maintain associations with the vertically transmitted fungal endophyte Epichloë festucae. A high prevalence of infected host plants in semiarid grasslands suggests that this association could be mutualistic. We investigated if the Epichloë-endophyte affects the growth and nutrient content of F. rubra plants subjected to drought. Endophyte-infected (E+) and non-infected (E−) plants of two half-sib lines (PEN and RAB) were subjected to three water availability treatments. Shoot and root biomass, nutrient content, proline, phenolic compounds and fungal alkaloids were measured after the treatments. The effect of the endophyte on shoot and root biomass and dead leaves depended on the plant line. In the PEN line, E+ plants had a greater S:R ratio than E-, but the opposite occurred in RAB. In both plant lines and all water treatments, endophyte-infected plants had greater concentrations of N, P and Zn in shoots and Ca, Mg and Zn in roots than E- plants. On average, E+ plants contained in their shoots more P (62%), Zn (58%) and N (19%) than E- plants. While the proline in shoots increased in response to water stress, the endophyte did not affect this response. A multivariate analysis showed that endophyte status and plant line impose stronger differences in the performance of the plants than the water stress treatments. Furthermore, differences between PEN and RAB lines seemed to be greater in E- than in E+ plants, suggesting that E+ plants of both lines are more similar than those of their non-infected version. This is probably due to the endophyte producing a similar effect in both plant lines, such as the increase in N, P and Zn in shoots. The remarkable effect of the endophyte in the nutrient balance of the plants could help to explain the high prevalence of infected plants in natural grasslands.

Double-stranded RNA and virus-like particles in the grass endophyte Epichloë festucae

Two double-stranded ribonucleic acid (dsRNA) elements were detected in an isolate of the clavicipitaceous grass endophyte Epichloe festucae obtained from an asymptomatic Festuca rubra plant. No dsRNA was detected in three other isolates of E. festucae. The size of the two dsRNA elements isolated from mycelium was of 5.2 and 3.2 kbp. Hybridization experiments with a cDNA probe complementary to the 5.2 kbp dsRNA showed that there was no sequence similarity between the dsRNA elements. Isometric virus-like particles of about 50 nm in diameter were observed by electron microscopy in sucrose-gradient purified virus preparations from the isolate containing dsRNA. The 5.2 kbp dsRNA element was present in the partially purified virus preparations, suggesting that this element is encapsidated. There was no difference with respect to growth rate or colony morphology between the dsRNA containing and dsRNA-free isolates. The presence of dsRNA and virus-like particles suggests the presence of a mycovirus in the Vit5 strain of E. festucae.

Are endophytes an important link between airborne spores and allergen exposure

Grasses represent one of the Earth’s most common plant groups, and natural and cultivated habitats dominated by grasses cover about 40% of the land surface. In turn, each grass species hosts multiple fungal species which can behave as endophytes. An analysis of the endophytic taxa identified in surveys conducted in 14 grass species showed that some of the most frequent taxa on each grass were also present across several host grasses. These taxa were Alternaria, Epicoccum, Cladosporium, Fusarium, and a few others. A similar analysis of airborne fungi surveyed at 41 different locations throughout the world showed that some of the most geographically widespread, and most locally frequent airborne fungi belonged to the same genera that are dominant endophytes in grasses (i.e. Cladosporium, Alternaria, Fusarium, etc.). Therefore, airborne spores of genera that are ubiquitous in grasses are common worldwide and attain high atmospheric concentrations. In addition, spores of the above mentioned fungi are also important respiratory allergens. Direct observation indicates that saprobic colonization and sporulation of non-systemic grass endophytes could become unrestrained when their host plant tissue dies. Subsequently, when appropriate environmental conditions favour sporulation on grass host surfaces, the natural cycle for airborne conidia initiates, and large numbers of these conidia disperse as inoculum for new endophytic infections. Therefore, the cycle of endophytism may be an important link between climate, plant biology and public health.

Pathogenicity of endophytic entomopathogenic fungi to Ornithodoros erraticus and Ornithodoros moubata (Acari: Argasidae).

The argasid ticks O. erraticus and O. moubata are of great medical and veterinary importance because they are vectors of the African swine fever virus and several species of human relapsing fever borreliae. Biocontrol of these ticks using entomopathogenic fungi has not been previously reported. We examined the pathogenicity to different developmental stages of these two argasids of six strains of the fungal species Beauveria bassiana (strains Bb1764 and Bb2157), Lecanicillium lecanii (strains Ll586, Ll618 and Ll3047) and Tolypocladium cylindrosporum (strain Tc3398). Three strains, Bb1764, Bb2157, and Tc3398, caused in Spanish O. erraticus mean mortality rates between 34.4% and 62% in 14-28 days post-inoculation. Additionally, Bb2157 also induced in African O. moubata mean mortality rates of 31.9%. The remaining strains caused lower mortality rates and were not considered effective. This is the first study in which some strains of entomopathogenic fungi are found to be effective against argasid ticks of the genus Ornithodoros, and its results might justify further efforts towards the application of entomopathogenic fungal strains as anti-argasid biocontrol agents.