Production and Germination of Conidia of Trichoderma stromaticum, a Mycoparasite of Crinipellis perniciosa on Cacao

ABSTRACT Growth characteristics of the fungus Trichoderma stromaticum, a mycoparasite on the mycelium and fruiting bodies of Crinipellis perniciosa, the causal agent of witches'-broom disease of cacao, were evaluated under controlled environmental conditions. The ability of T. stromaticum to produce conidia and germinate on dry brooms was evaluated at three constant temperatures (20, 25, and 30 degrees C) and two constant relative humidities (75 and 100%). T. stromaticum produced abundant conidia on brooms at 100% relative humidity and incubation temperatures of 20 and 25 degrees C, but none at 30 degrees C. Sporulation of T. stromaticum was not observed at 75% relative humidity at any temperature. At 100% relative humidity and either at 20 or 25 degrees C, treatment of brooms with T. stromaticum suppressed C. perniciosa within 7 days. In contrast, at 30 degrees C, treatment with T. stromaticum had no effect on the pathogen in brooms maintained at either 75 or 100% relative humidity. Mycelium of C. perniciosa grew from brooms at all temperatures at 100% relative humidity. Conidial germination on broom tissue approximated 80% at temperatures from 20 to 30 degrees C. Results suggest that applying T. stromaticum under high-moisture conditions when the air temperature is below 30 degrees C may enhance the establishment of this mycoparasite in cacao plantations.     

Growth rates of, and mycelial interactions between, isolates of Crinipellis perniciosa from cocoa

Growth of 87 single-basidiospore isolates of Crinipellis perniciosa , derived from witches' brooms on cocoa in 10 localities throughout South America and the Caribbean, was examined at 25°C on five agar media (Czapek-Dox, prune extract, potato-dextrose, V8 and carboxymethylcellulose). Interactions (designated intermingling or mutually antagonistic) between paired mycelia of 64 isolates were determined on V8 plates.
Six somatic-compatibility groups were identified comprising isolates from; (1) Pichilingue and Rio Palenque in Ecuador, Chigorodo and Manizales in Colombia; (2) Sucua (Ecuador); (3) Manaus (Brazil); (4) Ouro Preto (Brazil); (5) Castanhal (Brazil); (6) Trinidad and Tobago. This geographical separation of isolates was supported by results of the growth tests; growth of isolates within each compatibility group differed from other groups on at least one of the five media.
Separation of isolates by these methods did not conflict with previous results from tests of pathogenicity and could be useful in selecting isolates for screening cocoa lines for resistance to C. perniciosa.     

Pathogenic variability amongst isolates of Crinipellis perniciosa from cocoa (Theobroma cacao)

Dry witches' brooms from cocoa were imported from various areas within Bolivia, Brazil, Colombia, Ecuador, Trinidad and Venezuela. Basidiocarps of Crinipellis perniciosa were induced to form on these brooms and seedlings of different types of cocoa were inoculated with basidiospores either on the hypocotyl or cotyledon bud. Host reactions were assessed mainly by recording stem base swelling and broom development at the cotyledon node (hypocotyl inoculations) or the extent of swelling and branching of shoots (cotyledon bud inoculations).
Results from 30 experiments indicated considerable diversity amongst isolates in inducing disease symptoms, but suggested that two groups or populations of C. perniciosa exist on cultivated cocoa. One group (A), comprising isolates from Bolivia and Pichilingue (Ecuador) and most isolates tested from Colombia, induced severe symptoms on cocoa with Scavina 6 as one parent; the other group (B), comprising isolates from Brazil, Trinidad and Venezuela, did not. Within these groupings variants could be further distinguished by particular host reactions. Isolates from Ecuador, especially from the Oriente, a centre of diversity for Theobroma cacao, showed a range of pathogenicity comparable to that found amongst isolates from cultivated cocoa over a much wider area.     

Random amplified polymorphic DNA (RAPD) analysis of Crinipellis perniciosa isolates from different hosts

DNA sequence polymorphisms among isolates of Crinipellis perniciosa , causing witches' broom disease in several genera and species in the Sterculiaceae, Solanaceae and Bixaceae, were revealed by random amplified polymorphic DNA (RAPD) banding patterns. Comparisons were made among isolates from Theobroma cacao (cultivars SCA6 and SVB), T. grandiflorum, T. obovatum, T. subincanum, Herrania sp. (all collected near Belém, Brazil, at three plots within two kilometres of each other), and Bixa orellana and Solanum rugosum (150 and 1440 km from Belém, respectively). Genetic similarities, as determined by Sorensen's coefficient ( Sc_ij ; fraction of shared bands), ranged from 1·00 ( T. cacao SCA6 and T. grandiflorum ) to 0·56 ( S. rugosum and T. subincanum). Results indicate that proximity is more important than host species for determining the genetic relationships among isolates, that there were two independent sources of inoculum at the site near Belém, and that the isolate from B. orellana is most closely related to the isolate from T. cacao SVB. Comparisons were also made among multispore cultures from different basidiocarps on the same T. cacao SVB broom, as well as monospore cultures from the same S. rugosum basidiocarp. Banding patterns were similar among basidiocarps on the same broom, but differences were detected among monospore cultures from the same basidiocarp.     

A novel method for producing basidiocarps of the cocoa pathogen Crinipellis perniciosa using a bran-vermiculite medium

A novel method for the production of basidiocarps from the mycelia ofCrinipellis perniciosa is described. This involved the colonization of a bran-vermiculite medium with pure culture of the fungus, prior to application of a peat-based casing. Basidiocarp production was induced by hanging the cultures in a cabinet where they were subjected to a daily cycle of wetting and drying. The method was successfully and reproducibly used to fruit isolates of all four known biotypes of the fungus within 10–16 weeks of inoculation.     

Comparison of Crinipellis perniciosa isolates from Brazil by ERIC repetitive element sequence-based PCR genomic fingerprinting

Fifty isolates of Crinipellis perniciosa originating from Theobroma cacao , Heteropterys acutifolia and Solanum lycocarpum , from six states within Brazil, were characterized through ERIC-PCR, representing the first application of this method for molecular characterization within C. perniciosa . Phenetic analysis of banding patterns revealed a separation of isolates on the basis of host of origin, with T. cacao -derived isolates showing only a 0·2 similarity level to a cluster comprising the isolates from H. acutifolia and S. lycocarpum . Considerable intraspecific variability was observed within C. perniciosa isolates from T. cacao , with distinct groups observed correlating with geographical origin. Given that a number of isolates from T. cacao from the Amazon region grouped with isolates from Bahia state, this work discusses the possibility that current C. perniciosa populations pathogenic on T. cacao in Bahia originated from the Amazon region, rather than from alternative host plants.     

Sensitivity of Crinipellis perniciosa to two triazole fungicides in vitro and their effect on development of the fungus in cocoa

Two triazole fungicides, hexaconazole and triadimenol, were evaluated for their effects on the growth of Crinipellis perniciosa in vitro, and for their ability to prevent broom formation on cocoa seedlings. Amounts of both fungicides required to reduce fungal growth, germ tube extension and basidiospore germination by 50% were found respectively to be less than 1, 10-150 and more than 200 mg/l. Reductions in germ tube length were associated with slower rates of cell growth, whereas growth of dikaryotic mycelium was reduced due to shorter cell lengths. Isolates of C. perniciosa showed different sensitivities to the fungicides, variation between collections from different localities being greater than between collections from the same locality. Hexaconazole gave good control of the disease applied as a spray to cocoa seedlings both before and after inoculation. Triadimenol showed good activity when used as a preinoculation soil drench and in some treatments where plants became infected no necrosis of host tissue was observed. Activity was greatly reduced by drench treatments applied 10 days after inoculation, and most infected plants formed necrotic cankers.     

Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar- S-methyl (ASM)

The benzothiadiazole compound acibenzolar- S -methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa , agent of witches' broom, and Verticillium dahliae , agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches' broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches' broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.     

Cocoa production in Ecuador in relation to dry-season escape from pod rot caused by Crinipellis perniciosa and Moniliophthora roreri

Cropping and disease patterns were observed over a period of 4 years in individual cocoa trees at three contrasting sites near Quevedo, Ecuador, with the aim of evaluating the importance of dry season production and the consequent avoidance of pod disease caused by Crinipellis perniciosa and Moniliophthora roreri. Distinction of the diseases on necrotic pods was aided by splitting open pods after removal from the tree. The year was divided into wet season (April to September) and dry season (October to March) harvest periods. The 3-month lag between the actual wet and dry seasons and these harvest periods allowed time for symptom development. Combined disease losses ranged from 19·0% to 62·1 % at the various sites. At all sites and in each year, more ripe healthy pods were harvested in the dry season harvest period than in the wet season harvest period. In general, pod losses caused by C, perniciosa were curtailed more sharply by the dry season than those caused by M. roreri , which at one site caused as much damage in the dry season as in the wet season. The proportion of the annual production falling in the dry season harvest period varied among sites and between years at a given site. A comparison of trees from two progenies growing side by side showed consistent differences in cropping patterns, and identified certain trees that were productive, yielded consistently in the dry season harvest period, and were little affected by M, roreri. Analysis of long-term rainfall data for the Quevedo region indicated that years which lack a normal dry season do occur, but only once every 10 years on average. Further exploitation of disease escape through selection and breeding appears to be both feasible and appropriate.     

An optimized screening method for identifying levels of resistance to Crinipellis perniciosa in cocoa (Theobroma cacao)

The effects of host age, leaf number, host type (clone or seedling), pathogen spore concentration and incubation time on inoculation with Crinipellis perniciosa (witches' broom disease of cocoa) were studied in greenhouse experiments using susceptible cocoa genotypes. Three methods of inoculation (agar-drop, water-drop and spray) were also tested. An optimized inoculation method was selected and tested for its repeatability as well as its ability to discriminate between various levels of resistance to C. perniciosa in cocoa. The optimized method (350 000 viable basidiospores per mL, 60 h incubation, agar-drop technique) produced 100% infection repeatedly, on both clonal and seedling plants of a susceptible genotype. Seedling age (2–12 months) and leaf number did not significantly affect the percentage of plants with symptoms or broom characteristics. This method discriminated effectively between the various levels of resistance in 14 cocoa genotypes and is recommended as an inoculation method to identify levels of resistance in germplasm collections. Symptom severity was shown to be a better measure of resistance than infection success.     

Factors influencing the production of basidiocarps and the deposition and germination of basidiospores of Crinipellis perniciosa, the causal fungus of witches' broom on cocoa (Theobroma cacao)

The production of basidiocarps by Crinipellis perniciosa on detached, dead witches'brooms from cocoa was assessed in relation to temperature, light, cocoa clone, age of broom and type of tissue, in cabinets with a daily cycle of 8 h wet and 16 h dry. More basidiocarps formed and matured at 20–25°C than at 25–30°C. In the latter regime the pilei were smaller and white, instead of the usual crimson colour, and the stipes were longer. No basidiocarps formed at 30–35°C. At 20–25°C. more basidiocarps formed and matured with light at 100 μE m^-2 s^-1 during the wet period than at 10 μE m^-2 s ^-1. Only one basidiocarp and five primordia developed on 20 brooms kept in the dark. Brooms from 10 cocoa clones at Pichilingue. Ecuador, differed in basidiocarp productivity. most basidiocarps forming on brooms from Seavina and least on ICS clones. The numbers of basidiocarps produced on brooms aged 1.2.3 or 4 months when detached from cocoa trees were similar but time to initiation of the first primordium differed considerably. More basidiocarps formed at nodes than internodes.
The discharge of basidiospores was optimal at 20–25°C and 80% RH: germination was optimal in water agar films. Neither process was dependent on light.     

Molecular Fingerprinting Suggests Two Primary Outbreaks of Witches' Broom Disease (Crinipellis perniciosa) of Theobroma cacao in Bahia, Brazil

Crinipellis perniciosa (Stahel) Singer is the causal agent of witches' broom disease in the Sterculiaceae, Solanaceae, and Bixaceae families. The disease is endemic to the Brazilian Amazon, and was first reported infecting Theobroma cacao (cocoa) in the State of Bahia, Brazil, in 1989. Random amplified polymorphic DNA (RAPD) analyses were performed on 46 isolates of C. perniciosa from cocoa that were collected from 15 counties in Bahia and the Brazilian Amazon. A total of 258 RAPD loci from 20 primers and three mixed primers were analyzed. Of these loci, 108 (42%) were polymorphic, with an average of 4.7 polymorphic loci per primer produced. Genetic similarities were estimated using Nei and Li's index and UPGMA clustering. Bootstrap analysis divided the phenogram into four significantly different clusters: two groups contained isolates from Ariquemes and from Ouro Preto, Rondônia, and the other two separated the isolates from Bahia into two major groups of C. perniciosa, classified as Group 1 (G1) and Group 2 (G2). The two groups of isolates from Bahia differed for their genetic similarity with the isolates from the Brazilian Amazon. The geographic distribution of the groups in Bahia suggests two independent focal points of introduction. Ongoing programs to screen for resistant cocoa genotypes should consider both groups of isolates.     

Effects of the fungus Crinipellis perniciosa, causal agent of witches' broom disease, on cell and tissue cultures of cocoa (Theobroma cacao L.)

The symptoms of witches' broom disease in cocoa, caused by the Basidiomycete fungus Crinipellis perniciosa , are pronounced swelling of the terminal and axillary buds followed in the long term by necrosis of this tissue. The direct effect of C. perniciosa on cocoa cells was examined under controlled conditions by growing primary and secondary phase cultures of the fungus separately and also with callus cultures and with cell suspensions. Both primary and secondary phase mycelium reduced growth of callus cultures by about 47% after one week compared with the controls. However, cell suspensions containing primary phase mycelium showed initial growth double that of the uninfected controls after 5 days, but then growth was reduced below that of the control and particularly when the primary phase became secondary phase mycelium. This change in fungal development coincided with the time that the cell culture reached the stationary growth stage. Cell cultures inoculated with stationary phase mycelium showed the same growth as the control after 5 days but then growth was reduced to 50% of the control after 19 days incubation and remained at this low level subsequently. The inhibitory effect of secondary phase mycelium was examined by incubating callus and cell suspensions with culture filtrate from liquid cultures of the secondary phase. Inclusion of 50% by volume of culture filtrate from the secondary phase in the growth medium for callus and cell suspensions, respectively, resulted in a reduction in growth of the plant tissue cultures. Addition of fungal culture filtrates also led to loss in potassium and loss of viability of cell suspensions and of isolated cells as represented by protoplasts. The necrotrophic mode of the secondary phase may be achieved through the production of phytotoxins acting on the host cell membrane.     

Identification and Pathogenicity of the Fungus Isolated from Butt Rot of Japanese Cypress, Chamaecyparis obtusa

  Butt rot of Japanese cypress, Chamaecyparis obtusa, in three 25-year-old stands with 178, 70 and 45 trees caused 69.1%, 75.7% and 76.1% of the respective stands to rot. In the field survey, corticioid basidiocarps with yellowish hymenia were sometimes observed on the cut ends of trunks and cut surface of stumps of C. obtusa and a few species of hardwoods. These basidiocarps were identified as Phlebia chrysocrea by morphological studies. The isolates were classified into six groups based on their colony characteristics. The basidiocarp isolates belonged to one of six groups. The isolates from C. obtusa proved to be P. chrysocrea by mating experiments. In inoculation experiments, P. chrysocrea was pathogenic to C. obtusa. Received 7 December 2000/ Accepted in revised form 16 July 2001     

Genetic Constitution and Pathogenicity of Lolium Isolates of Magnaporthe oryzae in Comparison with Host Species-Specific Pathotypes of the Blast Fungus

ABSTRACT Fungal isolates from gray leaf spot on perennial ryegrass (prg isolates) were characterized by DNA analyses, mating tests, and pathogenicity assays. All of the prg isolates were interfertile with Triticum isolates and clustered into the crop isolate group (CC group) on a dendrogram constructed from rDNA-internal transcribed spacer 2 sequences. Since the CC group corresponded to a newly proposed species, Magnaporthe oryzae, all of the prg isolates were designated M. oryzae. However, DNA fingerprinting with MGR586, MGR583, and Pot2 showed that the prg isolates are divided into two distinct populations, i.e., TALF isolates and WK isolates. The TALF isolates were virulent only on Lolium species, whereas the WK isolates were less specific, suggesting that gray leaf spot can be caused not only by Lolium-specific isolates but also by less specific isolates. We designated the TALF isolates as Lolium pathotype. The TALF isolates showed diverse karyotypes in spite of being uniform in DNA fingerprints, suggesting that theyare unstable in genome organization.     

Rapid Screening of Cacao Genotypes for Field Resistance to Phytophthora palmivora Using Leaves, Twigs and Roots

Black pod, caused by Phytophthora spp. is one of the most important diseases of cacao occurring worldwide. Losses due to black pod caused by P. palmivora are still moderate in Côte d'Ivoire but P. megakarya causes high losses in Ghana and other Central African countries. Variation in field attack has been observed between cacao genotypes, but evaluation of pod losses is unsuitable for obtaining rapid progress in breeding. Results of inoculation tests using young detached leaves, twigs and roots, obtained from field and nursery plants, are presented here and compared to field resistance of similar genotypes observed over a 10-year period. Nine different Upper Amazon Forastero genotypes were tested together with progenies obtained by crossing these with the susceptible check IFC5 (Amelonado genotype). Rank correlations between the early screening tests and the level of field attack were positive and mostly significant (r=0.58–0.95). The coefficient of correlation was slightly higher for leaves (r=0.88) and roots (r=0.89) than for twigs (r=0.76). Also, resistance of the different plant organs was correlated (r=0.6–0.9). Resistance of the Upper Amazon parents was well correlated with the resistance of their cross progenies (r=0.7–0.9), suggesting that resistance is highly heritable. Resistance of leaves and twigs from the nursery was better correlated with field resistance than resistance of leaves and twigs from the field, which might result from more uniform growing conditions in the nursery. Inoculation of leaves appears the most suitable early screening method for black pod resistance. Application of this test in breeding more resistant cacao cultivars is discussed.     

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches?? broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70?M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.     

Interaction of Moniliophthora perniciosa biotypes with Micro‐Tom tomato: a model system to investigate the witches' broom disease of Theobroma cacao

The miniature tomato (Solanum lycopersicum) cultivar Micro‐Tom (MT) has become an important platform to investigate plant–pathogen interactions. In the case of the witches' broom disease of Theobroma cacao (cacao), the existence of Moniliophthora perniciosa isolates pathogenic to Solanaceae (S‐biotype) may enable the use of MT to circumvent limitations of the cacao host, whereas the availability of a non‐infective cacao C‐biotype allows the evaluation of contrasting responses of MT. Infection of MT by the S‐biotype led to stem swelling and axillary shoot growth to form broom‐like symptoms similar to the biotrophic phase in cacao, but the infected tissues did not progress to necrosis. Conversely, inoculation with the C‐biotype did not cause typical symptoms, but reduced plant height, appearing as a non‐host interaction. Histopathological characterization of the S‐biotype infection of MT by light and electron microscopy indicated limited germ tube penetration, preferentially through wounds at the base of trichomes or actively through the epidermis. No intracellular mycelium was observed, corroborating the lack of the necrotrophic stage of the pathogen. The analysis of gene expression during the interaction between the S‐ or C‐biotype with MT indicated that expression of plant defence‐associated genes differs for kinetics and intensity between a compatible or incompatible M. perniciosa–MT interaction. The pattern of spore germination and low rate of mycelia penetration suggests that the S‐biotype is not a fully adapted tomato pathogen, but possibly a case of broken non‐host resistance, and evidence suggests the occurrence of a non‐host MT response against the C‐biotype.     

Canopy-microclimate effects on the antagonism between Trichoderma stromaticum and Moniliophthora perniciosa in shaded cacao

The collective impact of several environmental factors on the biocontrol activity of Trichoderma stromaticum ( Ts ) against Moniliophthora perniciosa ( Mp ), the cause of cacao witches' broom disease, was assessed under field conditions of shaded cacao ( Theobroma cacao ) in south-eastern Bahia, Brazil. Biocontrol experiments were performed adjacent to an automated weather station, with sensors and Ts -treated brooms placed at different canopy heights. Sporulation occurred at the same dates for all Ts isolates, but in different quantities. Broom moisture >30%, air temperature of approximately 23 ± 3°C, relative humidity >90%, solar radiation intensities <0·12 KW m⁻² and wind speed near zero were the key environmental parameters that preceded Ts sporulation events. A multiple logistic regression indicated that these weather variables combined were capable of distinguishing sporulation from non-sporulation events, with a significant effect of wind speed. Analyses of environmental factors at ground level indicated similar pre-sporulation conditions, with a soil moisture content above a threshold of 0·34 m³ m⁻³ preceding all sporulation events. The sporulation of five selected Ts isolates was compared at four different canopy heights. Isolates responded differently to weather variation in terms of sporulation and antagonism to Mp at different canopy levels, indicating that different microclimates are established along the vertical profile of a shaded cacao plantation. The potential of these findings for development of predictive mathematical models and disease-management approaches is discussed.