Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields

Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real‐time PCR technique to detect the latent presence of C. acutatum in cold‐stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within‐field spread of the pathogen on strawberry leaves, with a within‐row dispersal distance up to at least 1·75 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real‐time PCR method, was significantly (P < 0·05) correlated with the incidence of fruit rot at harvest and post‐harvest (r = 0·56–0·66). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field.     

Tracing Latent Infection of Colletotrichum acutatum on Strawberry by PCR

Colletotrichum acutatum, a quarantine organism on strawberries in the EU, was found in Finland for the first time in 2000. Concern about rapid, unnoticeable spread of this pathogen has necessitated studies to find methods with which the quiescent fungus infection can be detected in imported, cold-stored strawberry plant material. Successful detection of C. acutatum in strawberry tissues by polymerase chain reaction (PCR) is dependent on the method of DNA extraction used. Good-quality nucleic acid, free of PCR inhibitors, was successfully prepared by slightly modifying the DNA extraction method of a commercially available kit. Species-specific primers, previously described in the literature, were successfully used in the PCR reaction. C. acutatum was detected by PCR both on symptomatic and asymptomatic plant parts and in artificially and naturally infected strawberry tissues. Positive PCR results were obtained from ripe and unripe berries, runners, petioles and different parts of crowns. The data demonstrate that the PCR technique can be used to detect C. acutatum in strawberry tissue even in plant parts that do not show visible symptoms.     

Phylogenetic relationships and genome organisation of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> causing anthracnose in strawberry

Colletotrichum acutatum is a major plant pathogen which infects a broad range of host plants. Extensive research has been carried out on C. acutatum populations affecting various hosts in different geographical locations, showing a considerable genotypic and phenotypic diversity. Anthracnose, caused by Colletotrichum spp., is the major disease of cultivated strawberry, Fragaria x ananassa. In the present study, the phylogenetic relationships within a worldwide sample of fifty-two C. acutatum isolates collected from different strawberry cultivars have been established, by using ITS sequence analyses. Twenty-nine isolates clustered in the molecular group A2, in which seventeen out of eighteen Spanish isolates were included; this may indicate that the group A2 is the key group in Spain. The molecular polymorphism among C. acutatum isolates was determined by southern-blot hybridisation using a telomeric DNA probe. Results indicated that the minimum number of estimated chromosomes ranges between six and nine. The molecular characterisation of C. acutatum isolates was completed using the Pulsed-Field Gel Electrophoresis (PFGE) technique that resolved from six to nine chromosomal bands, this number being coincident with the number of chromosomes obtained by telomeric fingerprinting. The minimum total genome size was estimated to range from 29 to 36 Mb. Comparison of karyotypes patterns and southern-blot analysis demonstrated a high level of molecular polymorphism among C. acutatum isolates from different origins.     

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.     

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。     

Two subpopulations of Colletotrichum acutatum are responsible for anthracnose in strawberry and leatherleaf fern in Costa Rica

Strawberry, Fragaria × ananassa, and leatherleaf fern, Rumohra adiantiformis, are two important crops in Costa Rica. One of the most severe diseases affecting these crops is anthracnose, caused by members of the fungal genus, Colletotrichum (teleomorph; Glomerella). Eighty single-spore isolates from strawberry and leatherleaf fern were identified as Colletotrichum acutatum by species-specific PCR, and were further characterised by Universally Primed PCR (UP-PCR) fingerprinting analysis, and sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Morphological differences, genotypic variation revealed by UP-PCR fingerprinting analysis, and a single sequence polymorphism within the ITS2 region were found between the isolates from strawberry and leatherleaf fern, respectively. The UPGMA cluster analysis of the fingerprints clearly separated the isolates derived from strawberry and leatherleaf fern into two different clusters. Pathogenicity assays on detached strawberry fruits confirmed the apparent difference between the two groups of isolates. It is therefore suggested that the pathogens responsible for strawberry anthracnose fruit rot and leatherleaf fern anthracnose in Costa Rica, belong to two distinct subpopulations of C. acutatum.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Simple diagnosis using ethanol immersion of strawberry plants with latent infection by Colletotrichum acutatum, Dendrophoma obscurans, and Fusarium oxysporum f. sp. fragariae

Simple diagnosis by ethanol immersion (SDEI) to detect Glomerella cingulata was used to detect three other fungi that also cause latent infection of strawberry plants. Signs on strawberry leaves with asymptomatic latent infection by Colletotrichum acutatum became visible using SDEI. Salmon-pink conidial masses were produced in the acervuli on the treated leaves 5 days after incubation at 28°C. In the case of Dendrophoma obscurans, pycnidia with amber conidial masses formed 5 days after incubation at 28°C. The pycnidia were observed mainly on the ribs, and conidial masses exuded from the ostiole. These macroscopic conidial masses were similar to those of G. cingulata and C. acutatum. When water was dripped onto a lesion caused by D. obscurans, the pycnidia exuded white filamentous conidial masses, making the distinction of D. obscurans from G. cingulata or C. acutatum. On petioles with latent infection by Fusarium oxysporum f. sp. fragariae, white aerial hyphae grew out from the vascular tissues on the cut surface 3 days after incubation at 28°C and were easily observed by eye or with a loupe. Thus, SDEI was also useful for diagnosing latent infection of strawberry plants by C. acutatum, D. obscurans, and F. oxysporum f. sp. fragariae.     

Comparison of six inoculation techniques withColletotrichum acutatum on cold stored strawberry plants and screening for resistance to this fungus in French strawberry collections

Six inoculation techniques were compared for their ability to evaluate resistance toColletotrichum acutatum of five strawberry cultivars. Inoculation by dipping the whole cold stored plants in a suspension of conidia adjusted to 2.10⁶ conidia ml^–1 made it possible to screen cultivars resistant to crown rot at 28 days after inoculation. Using the dipping technique, 44 strawberry cultivars were evaluated for their resistance to one strain ofC. acutatum, 1267b. Twelve of them did not show wilt symptoms and could be classified as resistant. When another strain ofC. acutatum, 494a, was inoculated to seven cultivars, all of them including Dover, resistant to 1267b, showed wilt symptoms. This result showed the importance of investigations on genotype × isolate interactions to conduct an efficient breeding programme for screening resistance toC. acutatum.     

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.     

Formation of highly branched hyphae by Colletotrichum acutatum within the fruit cuticles of Capsicum spp.

This study showed that Colletotrichum acutatum penetrates the cuticle layer of Capsicum spp. fruits by forming a previously uncharacterized structure from appressoria. This unusual structure was localized in the cuticle layer. The structure, formed within 24 h post‐inoculation (hpi), was a highly branched, well‐differentiated hypha which penetrated the epidermal cell at 72 hpi. The novel structure, with abnormally thick walls (about 250 nm), often formed multiple branches in the affected chilli pepper. This dendroid structure, probably required for penetration, was formed exclusively in the cuticle layer of chilli pepper fruits and was not found when C. acutatum was inoculated onto pepper petals, mango leaves, or fruits of tomato and aubergine. Colletotrichum acutatum produced similar dendroid structures within resistant chilli pepper fruits, but eventually these structures turned dark brown and no further infection in the epidermal cells occurred, implicating the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.     

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.     

Epidemiology,histopathology and aetiology of olive anthracnose caused by Colletotrichum acutatum and C. gloeosporioides in Portugal

Anthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003–2007 with 41% of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3–4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year‐to‐year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year‐round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre‐mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management.     

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.     

Epidemiology of apple bitter rot caused by <Emphasis Type="Italic">Colletotrichum acutatum sensu lato</Emphasis>

Apple bitter rot caused by Colletotrichum acutatum sensu lato results in fruit decay before and after harvest. We investigated the epidemiology of the disease in terms of conidial formation and dispersal as well as the change in susceptibility of fruits in Iwate, Japan. Conidia of C. acutatum were detected in rainwater collected from inside the tree canopy from May to August with peaks in production in mid-May to early June and mid- to late July. The first peak corresponded to the most conidia being produced on fruit scars, but the second peak was due to conidiation on mummified fruitlets and peduncles collected in July. Inoculation experiments revealed that fruits were susceptible to the pathogen between 20 and 90 days after petal fall and that immature fruits infected as early as 20 days after petal fall frequently developed lesions on the lower fruit half as growth progressed. These results suggest that C. acutatum sporulates on infested fruit scars to infect immature fruits, resulting in bitter rot and that the fungus also colonizes mummified fruitlets and peduncles, contributing to survival of the pathogen on fruit scars. Thus, infested fruit scars represent the primary source of inoculum.     

Management and cross-infectivity potential of Colletotrichum acutatum causing anthracnose on bell pepper in Florida

Early anthracnose caused by Colletotrichum acutatum has become an increasingly serious disease on green, unripe bell pepper fruit in Florida. This contrasts with earlier reports of anthracnose occurring on bell pepper primarily as a ripe-rot disease of mature, colored pepper fruit caused by Colletotrichum gloeosporioides. Management of anthracnose on green bell pepper fruit using fungicides and a commercial inducer of systemic acquired resistance, acibenzolar-S-methyl (ASM), was evaluated during three seasons. In two of the three trials, all the fungicides tested including azoxystrobin, fludioxonil + cyprodinil, mancozeb, famoxadone + cymoxanil, copper hydroxide, and ASM significantly increased the number of marketable fruit compared with control plants. These trials identified fungicides that could contribute to a successful pest management program on pepper for controlling anthracnose caused by C. acutatum. The cross-infectivity potential of C. acutatum was investigated on tomato and strawberry by in vitro and field inoculation. Anthracnose lesions formed readily on wound-inoculated detached fruits of all hosts in in vitro assays. Under field conditions, after inoculation, anthracnose lesions occurred on pepper fruit but no lesions of anthracnose were found on either ripe or unripe tomato or strawberry fruit in adjacent plots.     

Management of strawberry anthracnose by choice of irrigation system,mulching material and host resistance

We studied, by means of field experiments, the combined effects of irrigation system, mulching material and genetic resistance on the incidence of strawberry anthracnose caused by Colletotrichum acutatum. All trials involved artificial inoculation with fruits colonized by the pathogen. One experiment examined the effects of drip irrigation, overhead sprinkler irrigation, and localized low-pressure tape irrigation, and the effects of grass (Brachiaria sp.) and plastic (polyethylene) mulches, in a split plot, randomized complete block design with four replicates. Lower disease incidence, indicating lower pathogen dispersal, was verified with low-pressure tape and drip irrigation. Flower blight and fruit rot incidence in drip-irrigated plots was minimal (ca. 2% of the disease levels in the plots otherwise irrigated). Grass mulch reduced flower blight in the sprinkler system (up to 80%), but had no significant effect when disease levels were already low due to use of other irrigation systems. A second experiment compared the effects of grass mulch, pine (Pinus elliotti) mulch, and plastic mulch with overhead sprinkler irrigation on strawberry cultivars partially resistant (‘Dover’) and susceptible (‘Campinas’) to the disease in a factorial completely randomized design (CRD). Both organic mulches reduced flower blight and diseased fruit incidence by up to 76% compared to the plastic mulch treatment, especially in the early stages of the epidemics. ‘Dover’ had 70% lower incidence of flower blight than ‘Campinas’, and the effects of organic mulches were more pronounced in ‘Campinas’. A third experiment, with all three irrigation systems combined with inoculated and non-inoculated plots, estimated yield effects due to disease and irrigation system. All plots were covered with plastic mulch in a CRD with six treatments. This experiment clearly confirmed the higher flower blight and fruit rot incidences when sprinkler irrigation was used. In inoculated treatments, fruit yield was significantly reduced in tape-irrigated plots, and more so in sprinkler-irrigated plots. In drip-irrigated plots yield was reduced by only 1%. Overall, our results confirm the importance of cultural and genetic factors as valuable means of strawberry anthracnose management. Adoption of localized irrigation systems, the use of organic mulches and choice of a partially resistant cultivar significantly reduced disease levels in field plots. The importance of water splash for C. acutatum dispersal and its dependency on the soil mulch characteristics are the likely causes for the results observed.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.