Spatiotemporal characterization of citrus postbloom fruit drop in Brazil and its relationship to pathogen dispersal

Citrus postbloom fruit drop (PFD) is caused by Colletotrichum acutatum and C. gloeosporioides. These pathogens attack the flowers and cause premature fruit drop and the retention of fruit calyces. This study was designed to characterize the spatial and temporal dynamics of PFD in commercial citrus‐growing areas to better understand the disease spread. Experiments were carried out in three young orchards (500 trees each) in two municipalities in Sao Paulo State, Brazil. Symptoms of PFD on the flowers and presence of persistent calyces were assessed in each of three orchards for three years. Logistic, Gompertz and monomolecular models were fitted to the incidence data over time from the trees with symptoms. The spatial pattern of diseased trees was characterized by a dispersion index and by Taylor′s power law. An autologistic model was used for the spatiotemporal analysis. The logistic model provided the best fit to the disease incidence data, which had a fast progress rate of 0·53 per day. During the early epidemic of PFD, the spatial pattern of diseased trees was random, which suggested that inoculum spread was due to mechanisms other than rain splash. As the disease incidence increased (up to 12·6%), the spatial pattern of diseased trees became aggregated. The rapid rate of disease progress and the distribution of PFD suggest that dispersal of the pathogen is possibly related to a mechanism other than splash dispersal, which is more typical of other fruit diseases caused by Colletotrichum spp.     

Infection of citrus pollen grains by Colletotrichum acutatum

Postbloom fruit drop (PFD), an important disease caused by Colletotrichum spp., affects citrus yields in Brazil. PFD is characterised by the presence of necrotic lesions on the petals and stigmas of citrus flowers and by the subsequent abscission of young fruit. PFD epidemics have high disease progress rates, which is unusual for a pathogen that produces acervuli and is dispersed by rain. It is possible that other dispersal agents, such as insects and pollen, are involved in the spread of this disease. The objective of this work was to test whether citrus pollen grains can be colonised by Colletotrichum acutatum. Studies using light and electron microscopy showed that the pollen of Citrus sinensis can be infected by C. acutatum. This pathogen can penetrate and colonise citrus pollen grains 24 h after inoculation with the pathogen. The germ tube of conidia either penetrates the pollen sporodermis directly or passes through pollen germ pores. A single hypha can colonise more than one pollen grain. On the surface of the stigma, conidium formation can be observed. This study shows that Citrus sinensis pollen may, in fact, play a role in the spread of C. acutatum in citrus orchards.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

Molecular detection and quantification of Colletotrichum abscissum in sweet orange propagative material

Postbloom fruit drop disease (PFD) has caused serious impacts on citrus production in the Americas, occurring sporadically and suddenly during rain in the flowering period. In São Paulo State, Brazil, Colletotrichum abscissum is the causal agent responsible for over 80% of the disease incidence. Pathogen dispersal over long distances and the origin of primary inoculum are still unclear for PFD epidemics. We tested the hypothesis that citrus propagation material can harbour C. abscissum DNA by quantifying it in leaves of budwood increase block trees (BIB) and young citrus plants (YP) using multiplex quantitative PCR (qPCR). C. abscissum DNA was detected in all citrus nurseries, regardless of the type of propagative material, the sweet orange variety, or the nursery location. Overall, 73.4% of all samples from citrus nurseries have DNA from the pathogen, with a detection limit of 10 conidia. The average of 155 conidia found in YP was higher than the conidia observed on leaf samples from BIB (p = 0.03), although leaf samples from cultivars Valencia and Pera did not differ significantly, with means around 127 and 118 conidia, respectively (p = 0.75). This is the first molecular detection of C. abscissum in citrus propagative material. The multiplex qPCR assay may be used as a protocol for an accurate diagnosis of C. abscissum in citrus propagative material, may assist a better understanding of the pathogen dispersal over long distances, and may be used for further studies involving the quantification of C. abscissum in citrus orchards.     

Benomyl sensitivity assays and species-specific PCR reactions highlight association of two <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> types and <Emphasis Type="Italic">C. acutatum</Emphasis> with rumple disease on Primofiori lemons

Rumple is a serious peel collapse of Primofiori lemons in the southeast of Spain with an unresolved aetiology. Symptoms typically occur on fruits at ripening under wet conditions as dark sunken lesions producing premature fruit drop and damaged fruits unacceptable for fresh commercialization. A total of 16 Colletotrichum spp. isolates established from rumple-affected lemons collected during the autumn of 2007 from two different orchards were characterized by molecular and phenotypic assays and compared with reference isolates. Species-specific PCR reactions using β-tubulin 2 nucleotide sequences showed Colletotrichum gloeosporioides to predominate (81.5%) with limited occurrence of C. acutatum (18.75%). Among the C. gloeosporioides isolates, five (38.5%) showed benomyl resistance and eight (61.5%) were highly sensitive to the fungicide. The limited occurrence of C. acutatum could be related to factors such as the presence of both species on the same fruit, unfavourable meteorological conditions and low disease incidence. This work reveals an association of C. gloeosporioides and C. acutatum isolates with rumple disease of lemons and expands the range of C. acutatum on citrus.     

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of São Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in São Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of São Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.     

Most Colletotrichum species associated with tree tomato (Solanum betaceum) and mango (Mangifera indica) crops are not host‐specific

An important constraint for crop production in Colombia is the high incidence of anthracnose caused by Colletotrichum species. Although several studies have focused on these fungi, the relationship between the different fungal species within the genus and their hosts and whether they display any host preference or host specificity has yet to be examined. In Colombia, diseases caused by Colletotrichum species are particularly severe in mango (Mangifera indica) and tree tomato (Solanum betaceum) crops. In a previous investigation, the Colletotrichum phylogenetic species attacking these crops were identified. The present study aimed to determine whether isolates collected from tree tomato and mango showed host preference or host specificity by assessing aggressiveness, spore density, latent period, and fitness of each strain on the two hosts. In the departments of Cundinamarca and Tolima, Colombia, isolates were collected from plants that presented typical anthracnose symptoms and were identified as C. acutatum, C. asianum, C. boninense, C. gloeosporioides, C. tamarilloi and C. theobromicola. Inoculation of conidia of each isolate onto both hosts showed isolates had no host preference and only the C. gloeosporioides isolate showed host specificity. However, in general, isolates produced a higher spore density when inoculated on the alternate host, which may indicate a difference in the degree of adaptation to each host. Statistical analyses of the assessed parameter values revealed that isolates use different infection strategies when infecting each host. In light of these results, the implications of using quantitative estimations of fitness when studying fungal pathogens are discussed.     

Phenotypic and genetic characterization of Colletotrichum isolates from Belgian strawberry fields

Colletotrichum isolates (457) were collected from strawberry plant tissues with and without typical anthracnose symptoms and from symptomless weeds in 19 Belgian strawberry fields. The isolates were characterized based on genetic, morphological and pathological features. Isolates were classified according to rDNA‐ITS sequencing: 97% of 211 representative isolates were C. acutatum, 2%C. gloeosporioides and 1%C. coccodes. The C. acutatum isolates belonged to the intraspecific groups A2 (33%), A3 (5%), A4 (50%), A5 (3%) and A7 (6%). Differences in spore morphology, growth rate and colony colour of a selection of 146 isolates confirmed the genetic grouping. Multiple Colletotrichum genotypes were detected in the same field. There was no association between the most common genotypes and geographic origin, presence or absence of symptoms, nor plant species or plant part. Representative Belgian Colletotrichum isolates were used in pathogenicity tests, together with European and American reference isolates. The C. acutatum A2 and the Belgian C. gloeosporioides isolates were the most aggressive on fruits, followed by C. acutatum A3, A4, A5, A7 and C. coccodes isolates. When inoculated into crowns, C. acutatum A2, A5 and American C. gloeosporioides isolates were the most aggressive, followed by C. acutatum A3 isolates. The A4 and A7 isolates and all European C. gloeosporioides isolates were non‐pathogenic on crowns. These data indicate that an unusually diverse Colletotrichum population is present in Belgium. The traditional differentiation between C. acutatum and C. gloeosporioides as causal agents of fruit and crown rot, respectively, proved not to be valid in Belgian strawberry fields.     

Phylogenetic and morphological identification of Colletotrichum boninense: a novel causal agent of anthracnose in avocado

In recent years, anthracnose has become a significant disease affecting avocado fruit in the state of Michoacan, Mexico, where it significantly reduces fruit quality and commercial yield. Anthracnose has been assumed to involve Colletotrichum gloeosporioides and C. acutatum as causal agents. However, because of the increasing incidence of anthracnose, a more precise identification of the Colletotrichum spp. involved in this disease has become desirable. During the years 2004–2007, avocado fruits of different sizes exhibiting brown‐black and reddish spots on the pericarp and soft rot in the mesocarp, were gathered from orchards in nine counties. Fungal isolates were cultured on potato dextrose agar, and among these, 31 were selected for molecular, morphological and pathogenicity analyses. The molecular approaches used sequence typing of the internal transcribed spacer region and the partial nuclear large ribosomal subunit, allowing the unequivocal identification of C. gloeosporioides (71%), C. acutatum (16%) and C. boninense (13%). This last species has not been previously reported as being associated with anthracnose symptoms in avocado fruits anywhere in the world. Various morphological characteristics such as the size and shape of conidia were determined, as well as the conidial mass colour. Pathogenicity tests performed with all three species were conducted by inoculating healthy fruits. In each case, identical symptoms developed within 3 days of inoculation. Knowledge of the Colletotrichum populations in the Michoacan state, including the newly encountered avocado pathogen C. boninense, will facilitate further studies addressing the relationships between these Colletotrichum spp. and their avocado host.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

Studies on the spread of the olive scab pathogen,Spilocaea oleagina1

Investigations were carried out to study the occurrence and effectiveness of wind dispersal of conidia of the olive spot pathogen Spilocaea oleagina. Dissemination in absence of rain was confirmed both by collecting conidia at various distances from an inoculum source and by using trap plants. The dispersal gradient of conidia was very steep: 3–10% of leaves of olive seedlings placed 20 m from diseased trees became infected.     

Diversity and pathogenicity of Colletotrichum species isolated from soursop in Colombia

Anthracnose, caused by Colletotrichum species is a highly limiting disease for the production of the tropical fruit tree crop, soursop (Annona muricata L.). In this study, 83 single-spore isolates of Colletotrichum were obtained from diseased soursoup tissues and subjected to a species complex-specific PCR assay. The isolates were identified as C. gloeosporioides sensu lato (n?=?60), C. boninense s. lat. (n?=?22), or C. acutatum s. lat. (n?=?1). A subset of 21 selected isolates was identified to species level by means of a multi-locus phylogenetic analysis using sequences from the ITS region and partial sequences of the actin, β-tubulin-2, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase-1 genes. The multi-locus phylogenetic analysis resolved C. theobromicola, C. tropicale, C. siamense, and C. gloeosporioides sensu stricto in the C. gloeosporioides complex; C. karstii and one undetermined species in the C. boninense complex; as well as one undetermined species in the C. acutatum complex. Significant differences in anthracnose severity were observed between Colletotrichum species when tested for pathogenicity on attached twigs of soursop cv. Elita. Colletotrichum theobromicola and C. tropicale were associated with high and intermediate virulence, respectively, whereas the remaining species were associated with low virulence.     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.     

Improved method to induce sporulation of <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>, causal fungus of grape ripe rot

Colletotrichum gloeosporioides and C. acutatum are causal agents of grape ripe rot, but with available methods, sporulation of C. gloeosporioides on plate media has been unstable and inferior to that of C. acutatum. To facilitate studies on C. gloeosporioides, I developed an improved method to induce conidiation of this fungus. Isolates of C. gloeosporioides were pre-cultured in potato dextrose broth for 1 week, then pulverized in whole broth. The homogenate was then spread on diluted oatmeal agar (15–20% commercial oatmeal agar medium, 1.5% agar) plates. After the plates were cultured at 25°C under continuous light for another week, the C. gloeosporioides isolates sporulated stably on the plate medium.     

Identification of <Emphasis Type="Italic">Colletotrichum</Emphasis> species associated with anthracnose disease of coffee in Vietnam

Twenty-three isolates of Colletotrichum gloeosporioides, five isolates of C. acutatum, two isolates of C. capsici and six isolates of C. boninense associated with anthracnose disease on coffee (Coffea spp.) in Vietnam were identified based on morphology and DNA analysis. Phylogenetic analysis of DNA sequences from the internal transcribed spacer region of nuclear rDNA and a portion of mitochondrial small subunit rRNA were concordant and allowed good separation of the taxa. We found several Colletotrichum isolates of unknown species and their taxonomic position remains unresolved. The majority of Vietnamese isolates belonged to C. gloeosporioides and they grouped together with the coffee berry disease (CBD) fungus, C. kahawae. However, C. kahawae could be distinguished from the Vietnamese C. gloeosporioides isolates based on ammonium tartrate utilization, growth rate and pathogenicity. C. gloeosporioides isolates were more pathogenic on detached green berries than isolates of the other species, i.e. C. acutatum, C capsici and C. boninense. Some of the C. gloeosporioides isolates produced slightly sunken lesions on green berries resembling CBD symptoms but it did not destroy the bean. We did not find any evidence of the presence of C. kahawae in Vietnam.     

Histopathology of postbloom fruit drop caused by Colletotrichum acutatum in citrus flowers

Postbloom fruit drop (PFD) is caused by both Colletotrichum acutatum and C. gloeosporioides and is a potentially serious disease in citrus that occurs when flowering coincides with rainfall. The fungus incites necrotic lesions in petals and stigmas leading to premature fruit drop and reduced yield. The mechanisms of infection and survival of the causal agents remain to be fully elucidated. In the present study, we investigated the histopathology of PFD caused by C. acutatum in the petals and stigmas of sweet oranges using electron and light microscopy. In the petals, pathogen penetration occurred intra and intercellularly and also through the stomata, with intercellular penetration occurring most frequently. The distinct tissues of the petals were colonised, including the vascular system, particularly the xylem. Acervuli were observed on both sides of the petals. Although the fungus did not penetrate through the epidermal cells of the stigma, C. acutatum caused necrosis and an increase of phenolics in this tissue. A protective layer rich in lipophilic and phenolic compounds was formed under the necrotic area and crystals of oxalate were associated with the sites where the pathogen was present.     

Colletotrichum species associated with chili anthracnose in Australia

Phylogenetic relationships were determined for 45 Colletotrichum isolates causing anthracnose disease of chili in Queensland, Australia. Initial screening based on morphology, ITS and TUB2 genes resulted in a subset of 21 isolates being chosen for further taxonomic study. Isolates in the C. acutatum complex were analysed using partial sequences of six gene regions (ITS, GAPDH, ACT, CHS‐1, TUB2 and HIS3), and in the C. gloeosporioides complex were analysed using four gene regions (ITS, TUB2, ApMat and GS). Phylogenetic analysis delineated four Colletotrichum species including C. siamense, C. simmondsii, C. queenslandicum, C. truncatum and a new Colletotrichum species, described here as C. cairnsense sp. nov. This is the first reported association of C. queenslandicum, C. simmondsii and C. siamense with chili anthracnose in Australia; these species were previously associated with anthracnose on papaya and avocado. Furthermore, the dominant species causing anthracnose of chili in Southeast Asia, C. scovillei, was not detected in Australia. Inoculations on chili fruit confirmed the pathogenicity of C. cairnsense and the other four species in the development of chili anthracnose in Australia.     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。     

Molecular and physiological characterization of Arabidopsis–Colletotrichum gloeosporioides pathosystem

Anthracnose is a destructive disease that affects a wide range of crop plants especially in tropical and subtropical regions. Colletotrichum spp. are the major pathogens causing anthracnose. In this study, we collected and identified the pathogen from diseased samples of Stylosanthes, a major tropical forage crop. The ability of the pathogen to naturally infect Arabidopsis thaliana was examined. Sequence analysis of ITS, ACT, CHS, and GAPDH genes showed the pathogen to be Colletotrichum gloeosporioides sensu lato (s.l.), and this was supported further by morphological characterization of representative isolates. The disease symptoms and cellular infection process of aggressive isolates (DZ-19 and HK-04) and a weak isolate (CJ-04) were compared. DZ-19 and HK-04 caused more severe disease symptoms on both young seedlings and adult plants of Col-0 and Ws-2 ecotypes compared to CJ-04. Furthermore, the more aggressive isolates showed faster and earlier germination of conidia, formation of appressoria, and growth and development of hyphae during the infection. Genetic analysis of the defence response and expression profiling of defence marker genes demonstrated the involvement of MAP kinase, Ca²⁺-dependent protein kinase, salicylic acid, ethylene, and jasmonic acid pathways in the resistance against anthracnose. These results suggest that the Arabidopsis–Colletotrichum gloeosporioides pathosystem should provide a valuable tool for exploring the resistance mechanisms against this pathogen.