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.     

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.     

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.     

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.     

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.     

Analysis of resistance to witches’ broom disease (Moniliophthora perniciosa) in flower cushions of Theobroma cacao in a segregating population

This study evaluates resistance to witches’ broom disease in flower cushions of Theobroma cacao under field conditions. The aim was to determine optimal inoculation methods to evaluate the disease incidence using flower cushions in the field. A segregating mapping population of 580 trees (cultivar TSH 1188 × CCN 51) was analysed under two field conditions: high and low inoculum levels (in different years), corresponding respectively to trees with or without dried witches’ brooms hanging on the trees and producing basidiocarps. The number of newly formed cushion brooms in each tree was counted by the conventional method, and also the healthy and infected flower cushions in three 30 cm‐long regions along the trunk and the two main branches. The field inoculation methods discriminated between genotypes, with a 26% increase in disease incidence by Moniliophthora perniciosa at high inoculum. Two different segregation patterns were also observed: 27:27:9:1 under low, and 27:9:9:9:3:3:3:1 under high inoculum potential. It was also determined that at least 20 flower cushions were needed to accurately determine the percentage of infection. These methodologies allowed identification of the extreme phenotypes in this mapping population, and can therefore facilitate the detection of sources of resistance to witches’ broom disease.     

Biodiversity and biogeography of the cacao (Theobroma cacao) pathogen Moniliophthora roreri in tropical America

Moniliophthora roreri , the cause of moniliasis or frosty pod rot, occurs on the neotropical rainforest genera Theobroma and Herrania . While this basidiomycete has had devastating effects on the cacao tree ( T. cacao ) in tropical America, where it is confined, little is known of its biogeography and intraspecific genetic variability. Here, AFLP and ISSR profiles of 94 isolates of M. roreri from across its geographic range in Central/South America were analyzed. The study provided limited evidence to support the hypothesis that M. roreri is capable of sexual reproduction. The highest levels of genetic diversity occurred in Colombia and not in Ecuador as originally believed. The fungus was broadly divided into five genetic groups. Two of these have a wide geographic range: Bolívar group (north of Santander in Colombia, eastern Venezuela, peripheral Ecuador, Peru), and Co-West group (western Colombia, central Ecuador, Central America). The other groups are all apparently endemic to Colombia (Co-East and Co-Central groups) or north-western Ecuador ( Gileri group). We speculate that central/north-eastern Colombia may represent the centre of origin for M. roreri . Sequence data from the internal transcribed spacer region of the nuclear rDNA repeat were congruent with the AFLP/ISSR results, dividing M. roreri into two broad groups: the Orientalis group, comprising most isolates from the Co-East, Co-Central and Bolívar groups, and the Occidentalis group, comprising isolates from the Co-West and Gileri groups. The spread of M. roreri into new areas and countries mediated by human activity is discussed.     

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.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: interaction of cacao genotype, leaf age and duration of incubation

The effects were studied of four leaf development stages (LDS) and three durations of incubation (DI) on the accuracy of leaf-disc tests on eight cacao (cocoa) clones (C) for predicting field resistance to phytophthora pod rot caused by Phytophthora palmivora . The clones were known to possess different general combining abilities (GCA) for pod resistance in the field, evaluated monthly at harvest over a 9-year period. Disease severity (DS) was affected strongly by C, DI and LDS, with increasing levels of significance. Two- and three-way interaction effects were smaller than the clone effect, but still significant. Clone effects were most significant for LDS3 (i.e. leaves 50–60 days old) and for DI5 and DI7 (observations made 5 and 7 days after inoculation, respectively). Coefficients of rank correlation between DS and field results were significant for seven of the 12 treatments, with highest values obtained again for treatments LDS3/DI5 ( r = 0·87) and LSD3/DI7 ( r = 0·93). Pooling of data for different LDS and DI treatments did not further improve the correlation with field results. However, these correlations were improved (from an average of 0·74 to 0·88) when the GCA values for field resistance were based on weekly observations, carried out in one year, including losses of pods and cherelles. It was concluded that, when carried out in a standardized manner and under optimal conditions, the leaf-disc test may explain 75–90% of the genetic variation for field resistance of cacao genotypes to P. palmivora .     

Effect of genotype of cocoa (Theobroma cacao) on attractiveness to the mirid Sahlbergella singularis (Hemiptera: Miridae) in the laboratory

BACKGROUND: Mirids are a major constraint to cocoa growing in Africa. Cocoa breeding for tolerance/resistance could be effective in an integrated pest management system. Attractiveness is one aspect of tolerance/resistance, and decreasing the attractiveness of cocoa trees should be a good way of reducing damage. RESULTS: Small-scale laboratory tests were carried out in Cameroon to assess differences in the attractiveness to mirids of eleven cocoa genotypes. The genotypes were ranked according to their attractiveness score and a distance from a mean value. An analysis of variance was performed and revealed significant differences between cocoa genotypes (F=3.15, P<0.001). The cocoa genotype groupings revealed three major categories, with BE10 and SNK413 proving to be the most attractive. In contrast, five genotypes, IMC60, the Catongo Trinitario genotypes, Playa Alta2 (from Venezuela), SIC5 and SNK614, proved to be less attractive than the mean. Four genotypes (PA107, SNK619, UPA134 and T60/887) displayed similar attractiveness to the mean. CONCLUSION: The circular microtest offered the advantage of not needing a reference cocoa genotype. The least attractive clones, such as IMC60, were also the most tolerant in the field. Comparisons with the results of other studies are proposed.     

Effect of Humidity on Development of Tomato Powdery Mildew (Oidium lycopersici) in the Glasshouse

A series of experiments was carried out over four years in a glasshouse with computer control of humidity and temperature to investigate the effect of humidity on the development of tomato powdery mildew. Four relative humidities (RHs) (80%, 87%, 90% and 95%) at constant 19°C were maintained over an eight-week experimental period during the Autumn. Disease was greatest at 80% RH and was progressively less with increasing RH to a minimum level at 95% RH on both inoculated plants, introduced to act as initial infection sources, and on adjacent uninoculated plants. The results indicate that high humidities may decrease severity of this disease in the glasshouse and may help management of this disease in the future.     

Cultural, molecular and pathogenic variability of Mycosphaerella pinodes and Phoma medicaginis var. pinodella isolates from dried pea (Pisum sativum) in France

The characteristics of 50 isolates of Mycosphaerella pinodes and 17 isolates of Phoma medicaginis var. pinodella , originating from several regions of France where ascochyta blight is prevalent, were investigated using cultural, physiological, molecular and pathogenicity analyses. M. pinodes was distinguished from P. medicaginis var. pinodella on the basis of presence of pseudothecia, a higher proportion of larger, bicellular conidia, compared with the smaller, predominantly unicellular conidia of P. medicaginis var. pinodella , and a slower linear growth rate on agar under a 12-h light regime. RAPD analysis clearly distinguished the two species, which had low intraspecific variability. Although both species gave identical symptoms, they could be distinguished by their incubation period and aggressiveness, respectively, shorter and higher for M. pinodes . Virulence tests gave no definitive evidence for the existence of pathotypes among the M. pinodes isolates. Two unidentified isolates had similar characters to both M. pinodes and P. medicaginis var. pinodella in some features but were distinguished from them by their RAPD patterns.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: the effect of light intensity and time of day of leaf collection

Resistance of cacao leaves to Phytophthora palmivora was studied with regard to the time of leaf collection (morning, afternoon) and the degree of exposure of the leaves to light in the field (low, medium and high). The efficiency of leaf disc inoculations in predicting field resistance of nine clones was compared with that of detached and attached pod inoculations. Significant effects were observed, with leaves exposed to high light intensity and collected early in the afternoon showing highest susceptibility. The effect of time of leaf collection was reduced when leaves were stored overnight and leaf discs prepared and inoculated the following day, as compared to inoculations on the day of collection. Interactions between the main factors were significant, though less substantial than the clone effects. The most significant correlations with pod resistance ( r  = 0·70 to 0·97) were obtained for leaves collected early in the morning and exposed to intermediate shade conditions in the canopy. For other treatments, the correlations with pod resistance were still positive ( r  = 0·23 to 0·83) but often not significant. Pod inoculations in the laboratory were better correlated with field resistance ( r  = 0·92) than pod inoculations in the field ( r  = 0·72). Detached pod inoculations were also better correlated with leaf disc inoculations than those of attached pods. The results confirm the validity of laboratory inoculations of leaves and pods to assess field resistance to Phytophthora . Standardization of the leaf disc test is essential to obtain reliable results.     

广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

Molecular phylogenetic,morphological, and pathogenic analyses reveal a single clonal population of Septoria lycopersici with a narrower host range in Brazil

Septoria leaf spot, caused by Septoria lycopersici, is considered one of the most important diseases of tomato in Brazil. Despite its importance, the disease agent is still poorly studied. Septoria isolates collected from different production regions of Brazil were characterized by molecular, morphological, and pathogenic methods. A set of 104 isolates was sequenced for the DNA Tub, Cal, and EF1-α loci. Ten isolates were selected, according to geographical region of origin and type of leaf lesion (typical or atypical), for morphological characterization and for evaluation of aggressiveness on tomato cultivar Santa Clara. To evaluate the pathogen host range, cultivated and wild Solanaceae plants were inoculated with four selected isolates. The results showed that all isolates grouped with the type isolate of S. lycopersici in maximum likelihood and Bayesian inference trees. The isolates were morphologically similar. All isolates selected for pathogenicity testing on tomato were able to induce typical symptoms of the disease, but differed in their aggressiveness. A total of eight species of Solanaceae were also identified as potential alternative hosts for S. lycopersici. This information will provide a more accurate assessment of the risks involved with the introduction of new crops, especially of the genus Solanum, in areas where the species is already present. In addition, it will provide the basis for the establishment of more efficient methods in the management of Septoria leaf spot of tomatoes in natural conditions and in the different production systems.     

Microsatellite variability of sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides (Cyperaceae) in Kinki,Japan

Schoenoplectus juncoides is one of the most harmful weeds found in East Asian paddy fields. Recent emergence of biotypes that are resistant to the herbicide sulfonylurea (SU) has made weed control difficult. To examine the effect of the evolution of this herbicide resistance on genetic diversity within local populations, we investigated microsatellite variability within and among paddy field populations of S. juncoides in Kinki, Japan. In vivo assay of acetolactate synthase activity and root elongation assay in the presence of SU revealed that of 21 populations, five were sulfonylurea‐susceptible (SU‐S) and eight were completely sulfonylurea‐resistant (SU‐R). The remaining eight populations were a mixture of SU‐S and SU‐R individuals. The average gene diversity for SU‐R populations (H_S = 0.168) was lower than those for SU‐S (H_S = 0.256) and mixed (H_S = 0.209) populations, but the difference was not significant. This indicates that positive selection for SU‐R phenotype did not cause a genome‐wide reduction in genetic diversity. Genetic differentiation among S. juncoides populations was higher than that observed for most weed species studied previously. Although populations in neighbouring paddy fields showed a high level of differentiation, Bayesian clustering analyses suggested that some level of gene flow occurs among them and that the genetic exchange or colonisation between neighbouring populations could contribute to the geographical expansion of the resistant allele.     

Control of Schistosomiasis (Bilharziasis) in Brazil,Past, Present and Future

Abstract

Schistosoma mansoni was first recorded in Brazil in 1908, currently official estimates are that schistosomiasis is endemic in 1,000 municipalities and possibly ten million individuals are infected. Infection foci are rural communities and irrigated areas without sewage disposal facilities. In the past The Servicio Especial de Saúde Publica: The Servico Nacional de Malaria and later the Department of Rural Endemic Diseases (DNERu) attempted with some success to control the intermediate hosts, Biomphalarla spp., and improve sanitation and water supplies. Following examination of past policies DNERu in 1965 authorised the organisation of pilot projects for the control of schistosomiasis in 4 states: Pernambuco, Bahia, Minas Gerais, and Gaunabara. In 1970 the results of the pilot projects were evaluated and recommendations made regarding drug treatment and snail control for immediate use by the Superintendencia de Campanhas of the Federal Ministry of Health, involving the use of the drug Hycanthone, the molluscicide niclosamide (Bayluscide) and complimentary measures such as drainage and education in basic sanitation. In the future with the greater urbanisation of Brazil and increasing number of irrigation projects the incidence of the disease could increase. Improvements in sanitation, education and nutritional state will counteract this.     

Pathogenic and molecular diversity in highly virulent populations of the parasitic weed Orobanche cumana (sunflower broomrape) from Europe

The parasitic weed Orobanche cumana (sunflower broomrape) constrains sunflower production in eastern and southern Europe and in the Middle East. Although genetic resistance is the most effective control method, new parasite races evolve overcoming sunflower resistance. In this work, highly virulent populations of O. cumana were analysed for pathogenicity and genetic diversity. The virulence of 11 populations from Hungary, Romania, Spain and Turkey was assessed and compared after infection of sunflower inbred lines to differentiate races of the parasite under glasshouse conditions. Molecular diversity among and within 27 parasite populations was studied by RAPD‐PCR, UPGMA and amova analyses. Highly virulent race F was identified in Hungary, Spain and Turkey. The most virulent race (G) was also found in Turkey. The molecular analysis among highly virulent populations of O. cumana identified four molecular clusters, respectively, grouping populations from Central Spain, Hungary, South Spain and Turkey. The genetic homogeneity within parasite populations was confirmed, since no molecular divergences were found within them. This work constitutes the first geographical study of O. cumana together with pathogenicity and molecular traits inherent to each geographical group, and provides useful information for possible phylogenetic analyses of O. cumana. In addition, molecular markers associated with geographical origin could be developed and used as diagnostic tools to track new broomrape introductions into areas free of virulent races where they might represent a threat to sunflower production.     

Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.