Keith A. Holmes

Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri, in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases

The main constraint and continuing threat to cocoa production in Latin America is disease caused by two basidiomycete fungi belonging to the genus Crinipellis (Agaricales), both of which are currently on an invasive front. Classical biological control, in which coevolved natural enemies are considered to offer the most potential for sustainable control of invasive alien organisms, is investigated as a management strategy. Our initial approach has been to search for the suspected coevolved host (Theobroma gileri) of one of these pathogens, Crinipellis roreri, the causal agent of frosty pod rot, in the mesic forests of north-west Ecuador; to isolate the endophytes and mycoparasites associated with both host and pathogen; to screen selected fungi in the greenhouse and laboratory, in order to determine their biocontrol potential. Endophytes were isolated directly in the field from healthy tree boles and pods, and over 40 genera were recorded, mainly representing anamorphs of Hypocreales in the genera Acremonium, Clonostachys, Trichoderma and Verticillium, as well as basidiomycetes belonging to the Agaricales and Polyporales. Of the endophytic fungi tested, most can be inoculated into and recovered from asymptomatic cocoa seedlings. Twenty-eight mycoparasitic species were isolated from diseased pods and selected mycoparasites as well as endophytes were screened against C. roreri using the pre-colonised plate method. Consistently high mycoparasitism of the C. roreri pseudostroma was demonstrated, particularly by a complex of Clonostachys and Trichoderma species.

Taxonomy and biocontrol potential of a new species of Trichoderma from the Amazon basin of South America.

The new species Trichoderma ovalisporum is described and its biocontrol potential against Crinipellis species is analyzed. Using partial nuclear translation elongation factor 1α (EF-1α) and partial nuclear actin gene intron and exon sequences, T. ovalisporum is identified as a member of Trichoderma sect. Trichoderma and as a close relative of T. koningii-like taxa that have ellipsoidal to oblong, smooth conidia. Sequences of the internal transcribed spacer regions 1 and 2 (ITS) of the nuclear ribosomal gene cluster did not resolve the phylogeny of T. ovalisporum and its closest relatives. Trichoderma ovalisporum is morphologically similar to T. koningii, Hypocrea stilbohypoxyli, and three as yet undescribed Trichoderma taxa. It differs from its close relatives in having smaller, ovoidal conidia and in its fast rate of growth at 30 °C. Trichoderma ovalisporum was isolated twice: once from witches’ broom (Crinipellis perniciosa)-infected tissue of a liana (Banisteriopsis caapi, Malpighiaceae) collected in Ecuador. The second isolation was from the healthy bole of a mature tree of Theobroma grandiflorum (cupuaçu, Malvaceae) collected in Brazil (Pará). The liana isolate reinfected and was reisolated from meristematic tissues of seedlings of Theobroma cacao, and inhibited radial growth of the frosty pod rot pathogen (Crinipellis roreri) in vitro. It also persisted on the surface, and within the tissues, of cocoa pods in the field for at least 10 weeks.

Identification and characterization of an insect toxin protein, Bb70p, from the entomopathogenic fungus, Beauveria bassiana, using Galleria mellonella as a model system

An insect-toxic protein, Bb70p, was purified from Beauveria bassiana 70 using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. Bb70p has a high affinity for anion exchangers and 2D electrophoresis results revealed a single spot with a molecular weight of 35.5 kDa and an iso-electric point of ∼4.5. Bb70p remains active from 4 to 60°C, within a pH range of 4-10, but is more active in slightly acidic pH. A pure protein, Bb70p does not have any carbohydrate side chains. The protein caused high mortality by intra-haemocelic injection into Galleria mellonella with LD50 of 334.4 μg/g body weight and activates the phenol oxidase cascade. With a partial amino acid sequence comparison using the NCBI database, we showed no homology to known toxin proteins of entomopathogenic fungi. Thus, Bb70p appears to be an insect toxin protein, demonstrating novelty. Identification of this insect-toxic protein presents potential to enhance the virulence of B. bassiana through genetic manipulation.

Tackling black leaf streak disease and soil fertility constraints to enable the expansion of plantain production to grassland in the humid tropics

In Central Africa, plantain is traditionally grown after a forest fallow. Given increasing urban demand and a lack of forest fallows near urban centres, as well as poor roads and environmental concerns to reduce pressure on forests, research is needed to identify suitable shade, fertility management and cultivars to shift production of plantain to grasslands and to reduce losses to diseases such as black leaf streak disease (BLSD). Effects of light level (full, 67%, 33% light), and nitrogen (N)-amendment on BLSD-tolerant (FHIA-21) and BLSD-susceptible (Batard) cultivars planted on soil from paired grassland and forest sites were determined. BLSD and growth were monitored until 5 months after planting. Three months after planting, leaf area attacked on cultivar FHIA-21 was less than half that on Batard. Plants grown under 33% and 67% light had less leaf area attacked (2.9% and 4.6%, respectively) than those grown in full light (7.3%). Leaf area and dry matter (DM) were higher under shade and when grown on forest soils. Compared to growing BLSD-susceptible plantain on forested land under shade, a shift onto grasslands and a reduction in shade use is predicted to reduce yields. Using cultivar FHIA-21 may limit, but not eliminate, yield loss.