Identification of molecular markers linked to a gene conferring resistance to coffee berry disease (Colletotrichum kahawae) in Coffea arabica

Coffee berry disease (CBD) caused by Colletotrichum kahawae is a major constraint to Arabica coffee (Coffea arabica) production in Africa. One source of resistance to the disease is a natural interspecific hybrid between C. arabica and C. canephora and its derivatives. This study is aimed at deciphering the genetic basis of the host resistance and identification of molecular markers associated with it. CBD is a mature stage disease and in the absence of a mature mapping population, early detection of disease reaction phenotypes of mapping individuals is required. Two F₂ populations from crosses of cv. Catimor (resistant) and cv. SL28 (susceptible) were screened for resistance by a two step procedure. First, half of each population was screened 6 weeks after germination by inoculating hypocotyls with the pathogen. The surviving seedlings (G1) were considered to be resistant and were raised in a nursery together with the other unscreened halves (G2). Secondly, after one year, all the seedlings (G1 + G2) were screened by inoculation. Analysis of 57 microsatellites and 31 AFLP markers in 56 and 95 seedlings from G1 and G2, respectively, were performed. Eight AFLP and two microsatellites markers linked tightly to the resistant phenotype were identified and mapped to one unique chromosomal fragment introgressed from C. canephora. The gene conferring the resistance was localized within an 11 cM segment. It is concluded that the locus carries a major resistance gene designated Ck‐1, which is likely to be synonymous to the T gene described in previous studies.     

Distinguishing characteristics and vegetative compatibility of Colletotrichum kahawe in comparison with other related species from coffee

On the basis of pathogenicity tests on green berries or hypocotyls of coffee and by morphological and biochemical characteristics in culture, 31 isolates of Colletotrichum were classified into C. kahawe (24 isolates), C. gloeosporioides (six isolates) or C. acutatum (one isolate). Within these groups of isolates, vegetative compatibility groups (VCGs) were determined by complementation tests with mutants in the nitrate assimilation pathway. There were distinct incompatibility barriers between the three species. Among the C. gloeosporioides group, the three isolates tested were self-compatible but incompatible with each other. Within C. kahawe , 18 isolates were self-compatible and only one main VCG was detected. However, partial compatibility in C. kahawe was also indicated by variation in the intensity of heterokaryon formation between different pairs of isolates and between different types of mutant. The existence of only one VCG in C. kahawe is consistent with the low level of variation found in previous work on DNA polymorphism.     

Cytogenomic characterization of Colletotrichum kahawae,the causal agent of coffee berry disease,reveals diversity in minichromosome profiles and genome size expansion

Colletotrichum kahawae is an emerging fungal pathogen, which has recently undergone a speciation process from a generalistic ‘C. gloeosporioides species complex' background by acquiring the unique capacity to infect green coffee berries, thus causing coffee berry disease. This is a severe and widespread disease in Africa and an imminent threat to Arabica coffee cultivation in Asia and America, if the pathogen enters those continents. Genetic diversity within C. kahawae is low but notorious differences in pathogen aggressiveness have been described. This work characterized two cytogenomic traits (genome size and minichromosome profiles) of a collection of C. kahawae isolates, representing the breadth of its genetic diversity and distinct aggressiveness classes, along with closely related taxa. The results obtained constitute the first flow cytometry‐based genome size estimation in the genus Colletotrichum and show a c. 8 Mb genome size expansion between C. kahawae (79·5 Mb on average) and its closest relatives (71·3 Mb), corroborating evidence indicating that C. kahawae (i.e. the coffee berry disease pathogens) should remain as a distinct species. Results have also shown the presence of two to five minichromosomes in C. kahawae, suggesting a positive relationship between the number of minichromosomes and the level of aggressiveness of the different isolates analysed, while no correlation could be established between aggressiveness and whole genome size. Overall, these results may be the basis for the identification of pathogenicity/aggressiveness‐related factors in such minichromosomes, and may provide clues to the characterization of specific markers for aggressiveness classes.     

Variation among Colletotrichum gloeosporioides isolates from infected coffee berries at different locations in Vietnam

Isolates of Colletotrichum gloeosporioides associated with anthracnose disease on coffee berries in Vietnam were characterized by morphological and molecular methods. Random amplified polymorphic DNA (RAPD) and microsatellite-primered PCR (MP-PCR) analyses were employed to investigate the genetic variation among 38 and 51 isolates of C. gloeosporioides , respectively. According to both methods, the isolates mainly grouped in accordance with geographical origins. Higher genetic variation ( H  = 0·312 and 0·335) in the northern population of C. gloeosporioides than in the southern population ( H  = 0·261 and 0·186), according to the RAPD and MP-PCR markers, respectively, was indicative of a difference between the northern and southern populations. Moderate gene differentiation ( G st = 0·1) between populations from the north and the south was found. However, there was no differentiation between locations within the northern or southern populations, indicating significant gene flow. A four-gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam. The symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD (coffee berry disease; Colletotrichum kahawae ) isolates originating from Africa.     

Characteristics and distribution of Colletotrichum species in coffee plantations in Hainan,China

Anthracnose caused by Colletotrichum species is a serious disease on a range of economically important hosts. To determine the Colletotrichum species in coffee plantations in Hainan, China, 55 isolates were obtained from Coffea arabica (arabica) and C. canephora var. robusta (robusta) in five counties. Initially, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to measure fungal genetic diversity. Then a subset of 23 isolates was selected to represent the range of genetic diversity, varieties and geographic origin for further multilocus phylogenetic analyses. These isolates belong to eight known Colletotrichum species from three Colletotrichum species complexes, including gloeosporioides (C. endophytica, C. fructicola, C. ledongense, C. siamense and C. tropicale), boninense (C. karstii), gigasporum (C. gigasporum), and one singleton species (C. brevisporum). Of these, C. siamense was isolated in all sampled counties and C. fructicola was identified in three counties. The other six species were isolated only in one or two counties. Only C. siamense and C. fructicola were isolated from arabica, whereas all eight species were isolated from robusta. Occurrence of C. brevisporum, C. endophytica, C. ledongense and C. tropicale in coffee has not been reported previously. Pathogenicity tests showed that all eight species were pathogenic to coffee leaves and fruit. In vitro tests showed that Colletotrichum isolates from coffee in Hainan were most sensitive to prochloraz, less sensitive to carbendazim, propiconazole and difenoconazole, and least sensitive to myclobutanil.     

Disease complex in coffee involving Meloidogyne arabicida and Fusarium oxysporum

Coffee corky-root disease, also called corchosis, was first detected in 1974 in a small area of Costa Rica where the root-knot nematode Meloidogyne arabicida is the dominant species. An epidemiological study revealed a constant association between Meloidogyne spp. and Fusarium sp. in cases of corky root. No corky root appears to have been reported in association with Meloidogyne exigua , which is the prevalent root-knot nematode on coffee in Costa Rica. Fusarium spp. are often cited as components of disease complexes in association with nematodes. Combined inoculations using M. arabicida or M. exigua with Fusarium oxysporum under controlled conditions showed that only the combination with M. arabicida produced corky-root symptoms on Coffea arabica cvs Caturra or Catuai. Fusarium oxysporum alone was nonpathogenic. Meloidogyne exigua or M. arabicida alone caused galls and reduction in shoot height, but no corky-root symptoms. When cultivars susceptible and resistant to M. arabicida were studied under field conditions for 5 years, all the susceptible cultivars exhibited corky-root symptoms on 40–80% of their root systems. Cultivars that were resistant to M. arabicida but not to M. exigua showed no corky root. These observations lead to the conclusion that corky-root disease has a complex etiology, and emphasize the dominant role of M. arabicida as a predisposing agent to subsequent invasion by F. oxysporum . Consequently, genetic resistance to M. arabicida appears to provide an effective strategy against the disease.     

Effect of 2,4-dichlorophenoxyacetic acid applied as a herbicide on fruit shedding and coffee yield

The aim of this study was to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) applied as a herbicide at two timings after coffee blossoming, on fruit shedding from lower or upper plagiotropic branches on the canopy, as well as on crop yield. 2,4-D was sprayed between the rows on a 0.75 m-wide strip of land, starting on the border of canopy projection. Toxicity to the plants, final crop yield per plant and fruit shedding (FS) were assessed. The identical FS was observed for both application timings. Control plots showed an FS 13% lower than those where the highest 2,4-D dose was employed. FS was greater on the lower branches (46.8%) than on the upper ones (39.4%). This was probably due to injury to the lower plant canopy caused by the 2,4-D drift. Ripe or dried cherries and coffee bean yield per plant were not affected. It was concluded that despite the increased FS in the lower plant canopy, final coffee production was not significantly affected in response to 2,4-D application.     

Biochemical responses of coffee resistance against Meloidogyne exigua mediated by silicon

Coffea arabica cultivars Catuaí 44 and IAPAR 59, susceptible and resistant, respectively, to the root knot nematode Meloidogyne exigua, were grown in pots containing Si‐deficient soil amended with either calcium silicate (+Si) or calcium carbonate (?Si). There was an increase of 152 and 100%, respectively, in Si content of root tissue of cvs Catuaí 44 and IAPAR 59 in the +Si compared to the ?Si treatment, but no significant difference between Si treatments for calcium content. Plants, assessed 150 days after inoculation (d.a.i.) showed that the number of galls (NG) and number of eggs (NE) significantly decreased by 16·8 and 28·1% respectively, for susceptible cv. Catuaí 44 in the presence of Si, whilst both NG and NE were significantly lower for cv. IAPAR 59 compared to the susceptible cultivar regardless of Si treatments. In a separate experiment, biochemical assays were carried out 5 and 10 d.a.i. There was no significant difference between Si treatments and cultivars for concentration of total soluble phenolics. The concentration of lignin‐thioglycolic acid (LTGA) derivatives significantly increased by 11·5% in roots of nematode‐inoculated plants of susceptible cv. Catuaí supplied with Si. In roots of inoculated plants of resistant cv. IAPAR 59, the increase was 23 and 10%, respectively, for treatments with and without silicon. Peroxidase (POX), polyphenoloxidase (PPO) and phenylalanine ammonia lyase (PAL) activities significantly increased in roots of inoculated plants compared with roots of non‐inoculated plants, regardless of cultivar or Si treatment. In +Si treatments at 10 d.a.i., POX activity in roots of nematode‐inoculated plants of cvs Catuaí 44 and IAPAR 59 increased by 39·9 and 31·3%, respectively; PPO increased by 54·9 and 56·1%; and PAL activity was also higher at 26·6 and 62·9%. It was concluded that supplying Si to coffee plants increases root resistance against M. exigua by decreasing its reproductive capacity.     

Hypersensitive-like reaction conferred by the Mex-1 resistance gene against Meloidogyne exigua in coffee

The response of a susceptible coffee cultivar (Caturra) to infection by the root-knot nematode Meloidogyne exigua was compared histologically with that of cv. Iapar 59 possessing the recently identified Mex-1 resistance gene. The reproductive behaviour of the nematode was also compared in the two cultivars. Penetration and development in resistant plants were reduced in comparison with susceptible plants. Several cell features, including dark-stained cytoplasm and altered organelle structure, were observed in the resistant cultivar, indicating a hypersensitive-like (HR) response of the infested host cells. Features of giant cells were sometimes found beside necrotic-like areas, but the corresponding feeding sites were frequently associated with nematodes displaying abnormal shape. Six weeks after inoculation, root systems of cv. Caturra contained significantly more nematodes than those of cv. Iapar 59 (mean values 1574 and 41, respectively). The susceptible cultivar presented a minimum of 11 galls per plant, compared with only one or two galls per plant in the resistant cultivar. The findings are discussed in the context of plant–pathogen interactions.     

Distribution analyses of Meloidogyne spp. and Pratylenchus coffeae sensu lato in coffee plots in Costa Rica and Guatemala

The distribution of the plant-parasitic nematodes Pratylenchus coffeae sensu lato and Meloidogyne spp. were studied in two plots, one in Guatemala ( P. coffeae and M. paranaensis ) and the other in Costa Rica ( P. coffeae and M. exigua ). The quantity of nematodes per g fresh weight root were counted for each coffee tree sampled. The distributions were aggregated, and generally fitted well to negative binomial distributions. Population aggregation was greater when a smaller number of nematodes were involved, suggesting that initial colonization develops in foci. Analyses of the relationships between population levels of the species suggested that there was competition between Pratylenchus coffeae and Meloidogyne spp. This competition was expressed differently depending on the relative population density of the different species.     

Evidence for hyperparasitism of coffee rust (Hemileia vastatrix) by the entomogenous fungus, Lecanicillium lecanii, through a complex ecological web

The entomogenous fungus, Lecanicillium lecanii is hyperparasitic on Hemileia vastatrix , the cause of coffee leaf rust in the laboratory, and has frequently been observed attacking it in the field. The existence of a complex ecological web involving the spatially clustered mutualism of an ant ( Azteca instabilis ) with a scale insect ( Coccus viridis ), where the scale insect was infected by L. lecanii , prompted a search for a spatial correlation between the attack of L. lecanii on the scale insect and the incidence of rust in a commercial coffee crop. A weak but statistically significant effect of hyperparasitic control of coffee rust was observed on two distinct scales: in a 45-ha plot and on a scale of approximately 10 m. It was concluded that this effect was linked to an indirect effect of the ant–coccid mutualism, where L. lecanii was a parasite of the coccid.     

Role of rainfall in the development of coffee berry disease in Coffea arabica caused by Colletotrichum kahawae,in Cameroon

The development of coffee berry disease (CBD) epidemics (caused by Colletotrichum kahawae) in Cameroon was monitored over two successive years (2004 and 2005) on coffee trees protected from rainfall by transparent plastic sheets and on unprotected control trees. This work was done to assess how rain affected disease development when it did not fall directly onto the coffee trees and to determine the influence of primary inoculum on the severity of CBD. Weekly observations over the 2 years showed that there were 1·1% diseased berries on coffee trees completely protected from rainfall, compared with 45% diseased berries on unprotected coffee trees. Disease severity on unprotected trees during the 2 years of the experiment was estimated at 53% diseased berries, compared with 27% on trees only protected in the first year. These results confirmed rainfall as one of the key physical factors in the development of Arabica CBD. They also provided evidence of a subsequent effect of protecting coffee trees from rainfall in 2004 on the severity of CBD in 2005. This suggested some practices that might lead to very effective cultural control of CBD in regions where severe epidemics of the disease occur.     

亏缺灌溉与氮营养对小粒咖啡苗木生长及水分利用的影响

采用盆栽试验,设置了4个灌水水平：SI充分灌水（75%～85%θ_F）、DI₁轻度亏缺灌水（65%～75%θ_F）、DI₂中度亏缺灌水（55%～65%θ_F）和DI₃重度亏缺灌水（45%～55%θ_F）和4个施氮水平：高氮（N_H）、中氮（N_M）、低氮（N_L）、无氮（N_N）,研究了亏缺灌溉和氮营养对小粒咖啡苗木的生长及形态指标和水分利用效率的影响。结果表明,增加灌水或施氮能促进小粒咖啡生长,提高各形态指标（株高、茎粗、叶面积和枝条长度）。与无氮处理相比,施氮可使干物质累积总量提高10.88%～37.49%,水分利用效率提高24.73%～33.01%。与重度亏缺灌水处理相比,增加灌水可使干物质累积总量提高68.01%～141.90%,水分利用效率提高22.48%～40.89%。低氮充分灌水处理可提高小粒咖啡苗木的形态指标,获得较高的干物质累积和水分利用效率。     

云南西双版纳地区橡胶炭疽病病原鉴定

对来自西双版纳11个橡胶炭疽病菌菌株进行了鉴定。结果表明,引起西双版纳橡胶炭疽病的病原有2种,分别为:胶孢炭疽菌(Colletotrichum gloeosporioides)和尖孢炭疽菌(C.acutatum)。2个种的区别主要在于分生孢子的形态,菌落正反面的颜色,菌落的生长速度及对农药的敏感性等。     

Colletotrichum siamense is the main aetiological agent of anthracnose of avocado in south-eastern Brazil

Anthracnose caused by species of Colletotrichum is considered one of the main postharvest diseases for avocado. In this study, Colletotrichum isolates associated with avocado anthracnose, collected in different states of Brazil, were evaluated through phylogenetic analysis, morphological characterization, and pathogenicity assays. Moreover, the events during pathogen infection of avocados were examined by scanning electron microscopy. To assess the genetic diversity of 54 Colletotrichum isolates, partial sequence analysis of the gene gapdh was performed. According to the generated groupings and the geographical origins of isolates, a subset of 14 strains was selected for performing multilocus phylogeny analysis (using sequences of gapdh, act, tub2, and ApMat). Two species previously described were identified: C. siamense belonging to the C. gloeosporioides species complex and Colletotrichum karstii belonging to the C. boninense species complex. All Colletotrichum strains evaluated caused typical symptoms of anthracnose in avocado fruits. Conidia of the most virulent strain germinated between 6 and 12 hr after inoculation (hai). Penetration through wounds occurred 48 hai, tissue colonization occurred between 144 and 240 hai, and sporulation took place at 240 hai via the production of an acervulus, conidiophores, and conidia. The findings shed light on the aetiology of avocado anthracnose in Brazil and provide a better understanding of the infection process of this pathogen, which may assist in the development of disease management strategies.     

Foliar Aggressiveness of Phytophthora infestans in Three Potato Growing Regions in the Netherlands

Thirty-six isolates of Phytophthora infestans originating from three different potato growing regions in the Netherlands were tested for their aggressiveness to leaves of potato cultivar Bintje under controlled conditions. Measurements of latent period, maximal growth rate, infection efficiency and sporulation intensity were made and a composite aggressiveness index was calculated. Large variation in aggressiveness was present among isolates for each regional P. infestans population studied. The three populations differed significantly in latent period, but not for maximal growth rate and infection efficiency. Phenotypic variation existed for all components of aggressiveness and the aggressiveness index in P. infestans from each regional source. No association was found between mating type and aggressiveness. It is concluded that high levels of variation for aggressiveness are being generated and maintained through sexual reproduction in P. infestans strains from regional potato growing practices.     

茶褐枯病病原菌(Colletotrichum camelliae)生物学特性研究

为构建茶褐枯病病原菌(Colletotrichum camelliae)药剂筛选模型以及研究药剂的作用机理,本文对贵州省余庆县茶褐枯病病原菌(C.camelliae)进行病原生物学特性的研究。结果表明,菌株GZYQ-2016-01可在PDA、OA和MEA培养基上生长,其在PDA上的生长速率显著快于MEA与OA,在MEA和OA上无显著性差异。同时,菌株也可在PDB液体培养基中生长,其快速生长期为24~144h。产孢试验表明,菌株适宜在PDA培养基上产孢。碳素营养和氮素营养均影响菌丝的生长及色素形成,28℃是菌株适宜生长温度,菌株能较好地适应弱酸性条件。菌株在25℃条件下对茶树叶片的致病力显著强于20℃和30℃。     

日光温室茄茎腐病病原鉴定

1998～2000年连续 3年在甘肃省主要茄子栽培区大量采集茄子茎腐病病株和病土 ,经分离和致病性测定 ,表明引起当地茄茎腐病的主要病原为镰刀菌和疫霉菌。根据病原形态特征、培养性状以及致病性测定 ,确定其为尖镰孢菌 (Fusarium oxysporum)、黄色镰孢菌 (Fusarium culmorum)和寄生疫霉 (Phytophthora nicotianae)。两种镰刀菌生长发育最适温度均为20～30℃ ,最适pH为7.0;在光照条件下菌丝生长缓慢。     

灰霉菌(Botrytis cinerea)采后致病性研究

 灰霉菌(Botrytis cinerea)是引起葡萄采后病害的主要病原真菌之一。葡萄灰霉菌可在田间潜伏侵染,采后由健康果实携带进入销售市场,该菌的显著致病症状为果实软腐和脱落。灰霉菌与葡萄的其它采后致病菌,如链格孢菌(Alternaria alternata)、镰刀菌(Fusarium sp.)、芽枝霉(Cladosporium sp.)、青霉菌(Penicillium sp.)、黑曲霉(Aspergillus nigar)和粉红单端孢菌(Trichothecium roseum)相比,不仅表现出明显的潜伏侵染优势,而且具有较强的低温(4℃)条件下的致病优势。4℃低温下灰霉菌在寄主葡萄体外和体内分泌多聚半乳糖醛酸酶(PG)活力均显著高于以上各菌,而在25℃下无显著差异,这一结果与该2种温度下灰霉菌接种果实后的症状表现一致。