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Cocoa (Theobroma cacao) pod diseases cause tremendous losses, frequently eliminating over 80% of potential yield. Biological control has previously shown promise to supplement cultural control methods practiced by the majority of smallholders. Mixed Clonostachys inocula were most effective against multiple pathogens in Peru, but not in Costa Rica, where Clonostachys spp. seemed to antagonize Trichoderma spp. The objective of our study was to systematically investigate the mycoparasitic interactions between Clonostachys and Trichoderma species, in order to enable the design of mixed inocula in a rational manner. Specifically, we aimed to quantify dominance of one mycoparasite genus over the other using in vitro tests as well as field experiments. The compatibility of Peruvian and Costa Rican isolates of known efficacy was tested by pair-wise confrontations on water agar as well as two host-range assays, one of which mycoparasites were offered another mycoparasite as only host and one where conidia of a Clonostachys and a Trichoderma isolate were mixed at ratios ranging from 1:100 to 100:1 in order to determine dominance on the susceptible host, Phytophthora palmivora. A field study quantified survival and establishment of selected mixtures on surface-sterilized cocoa pods. Hyphal interactions and host-range studies coincided in identifying Clonostachys rosea as the most aggressive mycoparasite, closely followed by C. byssicola. Trichoderma spp. were least aggressive; instead, they were highly susceptible to Clonostachys spp. The outcome of interactions depended on the species used and occasionally even on the isolates confronting each other. The country of origin, however, was irrelevant. Parasitic growth of C. rosea was unaffected by Trichoderma harzianum, even at an inoculum ratio of 1:100. Parasitic growth of Trichoderma spp. progressively slowed as C. rosea concentrations rose, but the effect varied in strength with different isolates. When C. rosea and Trichoderma sp. were applied to cocoa pods in the field either separately or in combination at a ratio of 5:1, C. rosea dominated at the expense of Trichoderma sp., which was suppressed. At equal or ten times higher concentrations of T. harzianum, C. rosea was temporarily suppressed, but from two weeks after application, remained the dominant and persistent pod colonizer. Native Trichoderma spp. were poor pioneer colonizers of surface-sterilized pods and applied populations vanished within less than four weeks. Our results suggest that, although Clonostachys spp. antagonize Trichoderma spp., the incompatibility can be partly overcome by adjusting the inoculum ratio of mixed inocula in favor of Trichoderma spp. Therefore, mycoparasitism among several promising biocontrol candidates do not necessarily rule out their combination into a mixed inoculum, but can be addressed by formulating susceptible isolates at higher inoculum ratios. This, however, will not sustainably improve surface coverage by poorly colonizing Trichoderma spp. Therefore, optimal strains for the habitat and task are needed first.

Frosty pod rot (FPR), caused by Moniliophthora roreri, and black pod (BP), caused by Phytophthora spp., of cocoa (Theobroma cacao) cause combined pod losses of more than 80% in Panama. Biological control of both diseases appeared promising in Peru and is desired by certified organic producers in Panama. We evaluated both local and Peruvian fungal antagonists in participatory trials on smallholdings during two complete production cycles. Furthermore, we tested the influence of a 3% v/v cane molasses amendment on biocontrol efficacy, yield and population dynamics of mycoparasites on the cocoa pod. Significant variation was observed between the two years: FPR was more severe in the first year, BP in the second. FPR was significantly reduced by biocontrol agents (BCAs), but not by the molasses amendment. However, BCAs responded differently to molasses in both years. All BCAs reduced inoculum production by M. roreri with no consistent effect of molasses. BCAs had a lesser and more variable effect on BP, whereas molasses reduced BP slightly by increasing the efficacy of native antagonists. All BCAs and the molasses amendment enhanced the percentage of healthy pods. Molasses was beneficial to absolute yield, but only one inoculum improved yield significantly in the first year. Populations of a Peruvian Trichoderma asperellum isolate remained high for over two months after application to surface-sterilized pods. Molasses had no effect on establishment or survival of this antagonist or recolonization by any native mycoparasite. The reasons for enhanced biocontrol efficacy of the molasses formulation requires further investigation.

This article describes two field trials carried out at La Lola, Costa Rica, to assess control measures against frosty pod rot of cocoa (Theobroma cacao) caused by Moniliophthora (Crinipellis) roreri. In the first, factorial, trial the control agents were applied using motorised mistblowers (MMs) and hydraulic sprayers fitted with a narrow angle cone nozzle. There was an interaction between agents and application methods; together with previous application data for the most active fungicide (copper hydroxide), these trials indicate that best yields are achieved with sprays that maximise deposits on pods. We describe the droplet size spectra produced by a Stihl SR400 MM under a range of conditions because this has become the standard method of fungicide application in this series of trials at La Lola. The factor that had the largest effect on droplet size spectrum was the presence or the absence of a detachable baffle plate in front of the air-shear nozzle. In both trials described here, MMs were fitted with baffle plates, a formulation pump and restrictor transmitting 550 mL min - 1 to deliver an estimated equivalent of 190 L ha - 1 . Copper hydroxide as prophylactic applications at 1500 g a.i. ha - 1 have, to date, shown the most consistent (but incomplete) improvement in healthy pod yield. Use of copper fungicides may be cost effective when farm-gate cocoa prices exceed approximately $1.25 kg - 1 . In these trials, isolates of the hyper-parasitic fungi Clonostachys byssicola and Trichoderma asperellum and two off-patent triazole fungicides (bitertanol and triadimenol) made no significant improvement to healthy yields. The systemic oxathiin fungicide flutolanil, at a dosage of 300 g a.i. ha - 1 , appears to protect pods substantially at early stages but gives proportionately less control of M. roreri than copper at later stages of pod development.

Biocontrol agents of numerous insect pests and fungal pathogens exist but virtually nothing is known about their interaction if used simultaneously. Our objective was to investigate the compatibility of the entomopathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus, and the broad host-range mycoparasites Clonostachys spp., Trichoderma harzianum and Lecanicillium lecanii. In vitro host-range tests revealed that M. anisopliae was highly susceptible to all mycoparasites tested. B. bassiana was attacked by Clonostachys rosea, and P. fumosoroseus. was resistant to mycoparasites. M. anisopliae but not P. fumosoroseus killed nymphs of Bemisia tabaci in bioassays. B. bassiana and M. anisopliae proved lethal to Cosmopolites sordidus, Diatraea saccharalis and Sitophilus oryzae. Coapplication of mycoparasites with entomopathogens did not affect their biocontrol efficacy in vivo, although the reisolation success of entomopathogens could be significantly reduced, especially from smaller insect species. Trichoderma spp. were reisolated from mycoparasite-treated insects more frequently than C. rosea. The coapplication of the highly susceptible M. anisopliae generally enhanced mycoparasite recovery. Mycofungicide preparations caused some insect mortality but less than a copper hydroxide fungicide which is still permissible in organic agriculture. We concluded that the tested entomopathogens and mycoparasites are compatible elements of integrated pest management.

Many small (temporary) collections of fungi maintained by plant pathologists during their research receive inadequate attention to ensure stability. Maintaining collections of fungi in pure and viable conditions, minimising physiological and morphological changes is, however, a necessity. The objective of this study was to find preservation techniques for three Rosellinia isolates used in our plant pathogenic research. Various inert and nutritious carriers, solid as well as liquid, were used to test their suitability for conserving these Rosellinia isolates. Different cryoprotectants, cooling rates and thawing rates were tested to optimise liquid nitrogen storage procedures. Survival and/or growth rate were assessed over time. Rosellinia bunodes was the most difficult to store with survival not exceeding six to nine months using traditional storage methods in mineral oil and silica gel. Storage of Rosellinia necatrix and Rosellinia pepo was successful for periods up to at least 16 months in several carriers and for up to two years for R. necatrix in silica gel. Storage in liquid nitrogen proved no problem for R. necatrix or R. pepo with a 100% survival in all cases, although radial growth rates after recuperation were affected by cryoprotectant and thawing rates. Storage of R. bunodes was more difficult and survival as well as growth rates were affected by cryoprotectant and thawing rates. Cooling rates did not affect radial growth in any of the isolates. The results showed that development of a generalised procedure for storage of our Rosellinia species was not possible and successful storage protocols had to be developed for individual isolates.

Five native mycoparasitic strains of Clonostachys rosea and three of Trichoderma spp. were isolated from healthy cocoa tissue or basidiocarps of Crinipellis perniciosa using a baiting technique. They were compared singly or in combination with the commercial biocontrol agent Trichoderma virens (SoilGuard) for their potential to control three cocoa pod diseases: moniliasis, caused by Moniliophthora roreri; witches' broom, caused by C. perniciosa; and black pod, caused by Phytophthora palmivora. All isolates except Trichoderma T-1 inhibited basidiocarp formation of C. perniciosa under controlled conditions. The remaining isolates except Trichoderma T-3 reduced vegetative broom formation in a seedling bioassay. Clonostachys rosea G-3 and Trichoderma strains T-2 and T-3 significantly reduced symptoms caused by M. roreri in a seedling bioassay. Host-range studies identified P. palmivora as most susceptible to mycoparasitism and M. roreri as most resistant; C. perniciosa was intermediate. Different degrees of susceptibility were discovered at the pathogen strain level, especially for P. palmivora. However, broad host-range mycoparasites which attacked all three pathogen species were identified. Under field conditions, all selected treatments except a combination of C. rosea G-2 + G-3 reduced moniliasis, the main disease, significantly by 14.6–24.9% as compared with optimized, cultural control alone. No significant reduction of witches' broom or black pod was achieved but a combination of five C. rosea strains (G-1, G-2, G-3, G-4 + G-5) performed consistently best against all three diseases simultaneously. Yield increased by 16.7% and net returns by 24%. Control of moniliasis and yield were positively correlated to the number of mycoparasites in the inoculum. The results suggest that simultaneous biocontrol of the three major cocoa pod diseases with mycoparasite mixtures is highly promising. Future development strategies are discussed.

Thirty mycoparasites were detected using plates precolonized with pathogens of the crown-rot disease complex: Colletotrichum musae (6 strains), Fusarium moniliforme var. subglutinans (2 strains), F. moniliforme, Fusarium pallidoroseum, Botryodiplodia theobromae , and Nigrospora sphaerica (1 strain each). Dry leaves, dry flower residues, green leaves, and green peel were compared as mycoparasite sources. Dry leaves were the most prolific source, whereas green material collected in the canopy yielded the least number of isolates. Host species differed significantly in their suitability as bait for mycoparasites. C. musae was the best host for the isolation of most mycoparasites; fusaria were least colonized. No difference between strains of the same host species was detected during isolation. In contrast, in dual culture, different C. musae strains exhibited significant differences in susceptibility to mycoparasitism. The consequence of this finding for practical biocontrol is discussed. Thirteen aggressive antagonists, including Pythium sp., Trichoderma sp., Gliocladium sp., and Verticillium sp., were selected for further evaluation. Preliminary efficacy, host range, aggressiveness against survival structures (conidia and haustoria), and tolerance to fungicides presently used for crown-rot control were investigated. Eight mycoparasites had a reasonable number of desirable traits, but no single strain exhibited all features required for consistent biocontrol. It is suggested that future studies should consider combining two or three strains and/or to apply them with a reduced rate of a fungicide combined into the inoculum.

Two assessment methods for crown rot were compared: the widely used, categorical 0–5 scale and depth of rot penetration. Both were very highly correlated; the latter was more sensitive. The development of a bioassay for testing for crown rot pathogens and control methods is described. The rotting of green peel disks dipped in spore suspensions was significantly correlated with crown rot progress of whole clusters for both highly pathogenic C. musae and weakly pathogenic F. moniliforme isolates using inoculum concentrations covering a range of five orders of magnitude. The bioassay is specific to crown rot pathogens and detects efficient control measures. Because it is reliable, simple and rapid, it is suggested as a routine pre-screen for biocontrol agents against crown rot. The steps required for the establishment of a representative bioassay are explained and the final, optimised practical protocol is described.

This paper describes a factorial trial designed to investigate the delivery systems of fungal biological agents (Clonostachys rosea), applied as single strain or as strain mixture, in comparison with copper hydroxide as a chemical standard against Moniliophthora roreri in cocoa. Application techniques compared were motorised mistblowers fitted with rotary atomisers and hydraulic sprayers fitted with cone nozzles giving a narrow angle of spray. The third factor was the presence or absence of an emulsifiable rape-seed adjuvant oil in the tank mixture. Copper fungicide was the best agent for moniliasis control and resulted in the highest yield. Both the mycofungicides reduced sporulation of the pathogen. Net reduction of inoculum was best for the hydraulic sprayer-applied copper fungicide and the motorised mistblower-applied single-strain biocontrol agent. Overall, directional hydraulic sprays were found to be somewhat superior to the motorised mistblower technique, with its dissipated cone of spray. Addition of the adjuvant oil was the least important of the factors, but efficacy depended on the agent used, with a significant enhancement in the efficacy of copper with oil but a detrimental effect on C. rosea.

Single-strain biocontrol agents often look promising when tested against single-strain pathogens. When confronted with a biodiverse field population, however, biocontrol is inconsistent. This study implies that biodiversity of the crown rot pathogen Colletotrichum musae leads to strain discrimination by antagonists which results in variable biocontrol of the disease. Broad host-range mycoparasites of fungi of the crown rot disease complex of banana (C. musae, Fusarium moniliforme and Botryodiplodia theobromae) which attacked at least two of the pathogen genera, exhibited significant differences in aggression against different strain of C. musae, the main pathogen. Antagonists acted via several different mechanisms, i.e. parasitism, antibiosis or competition, simultaneously. The relative importance of each mechanism differed with the individual mycoparasites. Strain discrimination was correlated to differential susceptibility to one or more minor mechanism(s). When as many as four antagonists were combined into one inoclum, they complemented rather than antagonised each other. Biocontrol efficiency increased with the number of antagonist strains combined. Therefore, strain mixtures should be sought to control the corwn rot disease complex of banana.