Identification of Subpopulations of Colletotrichum acutatum and Epidemiology of Almond Anthracnose in California

ABSTRACT In recent years, almond anthracnose has developed into a major problem for the California almond industry. The identification of the causal pathogen as Colletotrichum acutatum was confirmed using species-specific primers and restriction fragment length polymorphisms of ribosomal DNA in comparative studies with isolates of C. acutatum from strawberry and C. gloeosporioides from citrus. Two distinct clonal subpopulations among the almond isolates of C. acutatum were identified. These two subpopulations differed in their colony appearance (pink versus gray cultures), conidial morphology, virulence in laboratory inoculation studies, temperature relationships for growth, and molecular fingerprints using random and simple-repeat primers in polymerase chain reactions. Both subpopulations were commonly isolated from the same orchard or even the same fruit. In other orchards, one subpopulation predominated over the other subpopulation. Using random, simple-repeat, and species-specific primers, isolates of the almond anthracnose pathogen from Israel were very similar to the California isolates that produce gray colonies. In addition to fruit, the pathogen was isolated from blighted blossoms, water-soaked or necrotic leaf lesions, symptomless peduncles, and spurs and wood from branches showing dieback symptoms, indicating that the amount of tissue that may be infected is more extensive than previously considered. Overwintering fruit mummies were identified as inoculum sources for early spring infections. Growth studies using almond kernels with different moisture contents indicated that postharvest damage of stored kernels likely originates from preharvest field infections.     

Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California

ABSTRACT The causal organism responsible for the recent outbreak of almond and peach anthracnose in California was identified and characterized as Colletotrichum acutatum. Isolates of C. acutatum from almond were found to be similar to California strawberry isolates and South Carolina peach and apple isolates of C. acutatum based on conidial morphology, temperature relationships, fungicide sensitivity, and polymerase chain reaction (PCR) methods using DNA species-specific primers. On almond, blossoms and immature or mature fruit were affected by the disease, causing direct losses of crop. On peach, the disease was observed only on mature fruit. Pathogenicity of almond and peach isolates of C. acutatum was demonstrated on wound- and nonwound-inoculated almond or peach fruit by fulfilling Koch's postulates. Conidial morphology of isolates was variable, depending on the medium or substrate used to culture the isolates. Isolates of C. acutatum from strawberry, almond, and peach were grouped together based on a similar response to temperature, with an optimal growth rate at 25 degrees C (generally less than 10 mm/day), whereas isolates of C. gloeosporioides from citrus and papaya had an optimal growth rate at 30 degrees C (generally greater than 10 mm/day). In fungicide disk assays, isolates of C. acutatum from strawberry, peach, and apple, as well as almond and peach isolates from California, were less sensitive to benomyl at 300, 600, or 1,200 mug/ml. In contrast, C. gloeosporioides isolates from citrus and papaya were very sensitive to benomyl at all concentrations evaluated. All isolates of both species were sensitive to captan (300, 600, or 1,200 mug/ml). Oligonucleotide primers were synthesized for C. acutatum, C. fragariae, or C. gloeosporioides using published DNA sequences from the internal transcribed spacer 1 region of ribosomal DNA. Thirty-two Colletotrichum isolates from almond fruit produced DNA products with a C. acutatum primer (CaInt-2) that matched products and approximate molecular weight of known C. acutatum isolates. No PCR products were produced with primers for C. gloeosporioides or C. fragariae. Isolates from citrus and papaya produced DNA products only with primers from C. gloeosporioides or C. fragariae. Thus, worldwide, anthracnose of almonds may be caused by either C. gloeosporioides, as previously reported, or by C. acutatum, as indicated in this study.     

PCR-based detection of Colletotrichum acutatum on strawberry

An oligonucleotide primer ( Ca Int 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum . PCR with primers Ca Int2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum . Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum . Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species-specific primer ( Ca Int2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.     

Hemibiotrophic infection of Pisum sativum by Colletotrichum truncatum

The infection of pea ( Pisum sativum ) by Colletotrichum truncatum was studied by light and electron microscopy. These investigations were facilitated by use of an Argenteum pea mutant, which has a readily detachable epidermis. Infection pegs emerging from appressoria penetrated epidermal cells directly. Large intracellular primary hyphae formed a dense stromatic mycelium confined within a single epidermal cell. Primary mycelia gave rise to thinner secondary hyphae which radiated into surrounding cells and caused extensive wall dissolution. Melanized sclerotia developed in the centre of chlorotic water-soaked lesions. Acervuli were not observed. Epidermal cells survived initial penetration by primary hyphae, as shown by their ability to plasmolyse and accumulate Neutral red, but all infected cells were dead when the secondary hyphae had formed. Six cultivars of pea were susceptible, but seven other legumes were resistant. A single isoform of polygalacturonase with a pI of 8·3 and apparent M _r of 40000 was purified from culture filtrates and the TV-terminal amino acid sequence determined. The relevance of the results to the taxonomy of C. truncatum and the relationships between infection process and host range are discussed.     

Occurrence and development of Colletotrichum acutatum on symptomless blueberry bushes

The anthracnose fungus Colletotrichum acutatum was detected in symptomless blueberry bushes ( Vaccinium spp.) in a Japanese blueberry field. Naturally diseased bushes and their apparently healthy neighbours were selected, and C. acutatum was isolated from the symptomless tissues of each bush from February 2000 to January 2002. Analysis of the diseased bushes during the dormant period revealed that the fungus was able to survive on symptomless tissues, such as shoot bark and bud scales. Furthermore, C. acutatum was consistently isolated from symptomless leaves and shoots of several surrounding symptomless bushes. Arbitrarily primed PCR (ap-PCR) analyses of the fungal isolates obtained from the diseased and symptomless bushes revealed that most C. acutatum isolates were genotypically identical, regardless of their origins. Inoculation tests using leaves of various blueberry cultivars suggested that the presence or absence of symptoms on each bush can not always be explained by differences in cultivar susceptibility, and other factors may be associated with the appearance of symptoms.     

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves

ABSTRACT The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.     

Colletotrichum acutatum and C. gloeosporioides Cause Anthracnose on Olives

Morphological and cultural features and restriction fragment length polymorphism analysis of ITS regions, including 5.8S rDNA, from 26 isolates of Colletotrichum species revealed that isolates from olive fruits, previously identified as C. gloeosporioides, belong to two taxa: C. acutatum and C. gloeosporioides. Comparison of both ITS sequence data with reference isolates confirmed the presence of both species in olives affected by anthracnose disease.     

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.     

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.     

Colletotrichum acutatum and Colletotrichum nymphaeae causing blossom blight and fruit anthracnose on olives in southern Brazil

European Journal of Plant Pathology - Olive (O. europaea L.) is an expanding crop in the south of Brazil. Blossom blight and typical anthracnose symptoms on fruit were observed in olive trees in an...     

Novel infection strategies of Colletotrichum acutatum on ripe blueberry fruit

The infection and colonization process of Colletotrichum acutatum on ripe blueberry fruit from two cultivars with different susceptibility to anthracnose were examined using light and confocal laser scanning microscopy. Ripe fruit from susceptible cv. Jersey and resistant cv. Elliott were drop-inoculated with a conidial suspension of C. acutatum, and epidermal peels were evaluated at selected times after inoculation and incubation. Results from pre-penetration studies demonstrated that there were significant differences in the rate of formation of melanized appressoria between the two cultivars, with the rate of formation being faster in the susceptible one. In both cultivars, penetration by the pathogen occurred via appressoria 48 h post-inoculation (hpi). However, in the susceptible cv. Jersey, C. acutatum then adopted an intracellular hemibiotrophic-like infection strategy, whereas in the resistant cv. Elliott subcuticular intramural-like infection occurred. In cv. Jersey by 108 hpi, intracellular growth of the pathogen led to the formation of numerous acervuli, with orange conidial masses. By 120 hpi, the conidial masses had coalesced covering the entire inoculated area. In cv. Elliott, acervuli were not seen until 144 hpi and contained few conidia. These results demonstrate for the first time the ability of C. acutatum to adopt a different infection and colonization strategy depending on the susceptibility of the host tissue being colonized.     

Effect of Rain Distribution Alteration on Splash Dispersal of Colletotrichum acutatum

ABSTRACT Splash dispersal of Colletotrichum acutatum conidia from infected strawberry fruit was assessed using a rain simulator to determine the properties of rain (e.g., intensity [millimeters/hour] or drop size distribution) most related to dissemination. Dispersal with a simulated rain corresponding to a natural rain of about 11 mm/h was compared with dispersal of three other simulated rains that had larger and smaller drop sizes, on average, than idealized natural rains. Splash droplets were collected in sheltered petri plates with a selective medium for Colletotrichum, and colonies formed from conidia entrained in the droplets were counted and used as the measure of dispersal. Colonies were mostly confined to a 27-cm radius from the source, and density of colonies decreased exponentially with the distance squared, as indicated by the fit of a diffusion-type model to the data. Splash dispersal was more affected by drop size distribution than rain intensity or other properties of the generated rains. That is, there was a direct positive relationship between total colonies over 61 min of rain for a circular area with a 72-cm radius (Sigma) and the mass (volume) median diameter of impacting drops (D(0)') for four rain-simulation treatments. In a separate study, strawberry fruit were exposed to the same four simulated rains at two distances from a point source and for two rain durations. Although the proportion of infected fruit (y) increased with time and decreased with distance, rain treatment did not significantly affect y, as predicted based on past work with a wide range of intensities of simulated rains.     

Genetic and Morphological Characterization of Colletotrichum acutatum Causing Anthracnose of Lupins

ABSTRACT Anthracnose, caused by Colletotrichum sp., is a serious problem of lupins (Lupinus spp.) worldwide. Morphological characters and molecular markers were used to characterize 43 Colletotrichum isolates from lupins, 8 isolates from other hosts, and 18 reference isolates representing related Colletotrichum spp., to assess the pathogen diversity and resolve its taxonomy. All lupin Colletotrichum isolates tested positive with C. acutatum-specific polymerase chain reaction (PCR) and did not test positive with C. gloeosporioides-specific PCR. Spore shape and colony diameter as well as insensitivity to benomyl grouped the lupin anthracnose isolates closer to C. acutatum than to C. gloeosporioides. Analysis of internal transcribed spacer (ITS) sequences of 57 Colletotrichum isolates grouped all lupin isolates with C. acutatum and distinct from C. gloeosporioides. Further, tub2 and his4 sequences revealed groups concordant with ITS, reducing the excessive dependence on the latter. Arbitrarily primed-PCR and amplified fragment length polymorphism analyses revealed intraspecific subgroups, but neither was useful to decipher species level relationships. ITS, tub2, and his4 results strongly support designating lupin anthracnose pathogen as C. acutatum or its subspecies. Most Colletotrichum isolates from lupins from worldwide locations are genetically homogeneous and form a distinct subgroup within C. acutatum. Present results also underline the potential of the C. acutatum-specific PCR for routine pathogen diagnosis.     

Heat treatment to control Colletotrichum acutatum on corms of Anemone coronaria

Anemone corms may be infected with the fungusColletotrichum acutatum, which under certain conditions causes leaf curl and leaf necrosis.A hot-water treatment (hwt) of infected corms for 1.5 h 47.5 °C or 1 h at 50 °C suppressed the disease almost completely. The period of application of hwt between harvest and planting time of corms did not influence the efficacy of hwt. Germination potential of different lots of untreated anemone corms varied between approximately 65% and 95%. Hot-water treatment for 1.5 h at 47.5 °C or 1 h at 50 °C reduced germination only slightly if corms of lots that germinated well without hwt were used. However, germination after hwt was severely reduced if lots with a poor germinating potential were used. Germination potential of hwt-treated corms was restored when they were stored for 4 days in moist vermiculite at 20 °C between hwt and planting. Drying of hwt-treated corms after storage in moist vermiculite reduced germination.A dry heat treatment of infected corms reduced disease incidence too, but results were inconsistent and germination of corms was considerably reduced when control of the fungus was optimal.     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

Strawberry Anthracnose: Histopathology of Colletotrichum acutatum and C. fragariae

ABSTRACT Ontogeny of the invasion process by Colletotrichum acutatum and C. fragariae was studied on petioles and stolons of the strawberry cultivar Chandler using light and electron microscopy. The invasion of host tissue by each fungal species was similar; however, each invasion event occurred more rapidly with C. fragariae than with C. acutatum. Following cuticular penetration via an appressorium, subsequent steps of invasion involved hyphal growth within the cuticle and within the cell walls of epidermal, subepidermal, and subtending cells. Both species of fungi began invasion with a brief biotrophic phase before entering an extended necrotrophic phase. Acervuli formed once the cortical tissue had been moderately disrupted and began with the development of a stroma just beneath the outer periclinal epidermal walls. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervulus maturation and remained minimal. Chitin distribution in walls of C. fragariae was visualized with gold-labeled wheat germ agglutinin. The outer layer of bilayered walls of conidia, germ tubes, and appressoria contained less chitin than unilayered hyphae in planta.     

Effects of a Cover Crop on Splash Dispersal of Colletotrichum acutatum Conidia

ABSTRACT A rain simulator, with generated rains of 11 and 30 mm/h, was used to determine the effect of a cover crop or intercrop on the splash dispersal of Colletotrichum acutatum conidia. Dispersal through sudangrass, which can be used as a 'living mulch', was tested at two planting densities (140 or 280 kg/ha) and two heights (5 and 20 cm) and compared with a control consisting of a bare soil. Dispersal of C. acutatum conidia was assessed by counting colonies formed from spore-bearing splash droplets deposited in sheltered petri plates containing a selective medium. Both a cover crop and rain intensity significantly affected splash dispersal as measured by the interpolated total number of colonies (denoted by Sigma) from 0 to 72 cm from the inoculum source and in a time span of 61 min of generated rain (P < 0.001). However, there was no significant interaction of cover crop and intensity (P > 0.90). Dispersal with a 30-mm/h rain was higher than dispersal with a 11-mm/h rain, and presence of a cover crop significantly reduced dispersal compared with bare soil (P < 0.001). Of the treatments with sudangrass, cover crop planting density did not affect dispersal overall, but there was greater spore dispersal with the taller sudangrass at the higher planting density, due in part to the higher rate of water splashing with the tall grass compared with the short grass. Spore deposition in the petri plates could be functionally related to distance and time using a diffusion-type model, and parameter estimates could be used to explain the effects of cover crop on Sigma. Although the relationship between cover crop properties and splash dispersal is complex, results show the potential beneficial effects of the cover crop on disease management.     

Inheritance of Resistance to Colletotrichum acutatum in Fragaria x ananassa

ABSTRACT Anthracnose, caused by Colletotrichum acutatum, is a major disease of the octoploid cultivated strawberry, Fragaria x ananassa The inheritance of high and intermediate level plant resistances to C. acutatum, pathogenicity group 2, was investigated in an 8 x 8 factorial design. A single dominant gene (Rca2) controlled the high-level resistance, although minor genes may also contribute to resistance in cultivars such as Belrubi. The intermediate level of resistance was quantitative and controlled by minor genes. Analysis of 26 genotypes and cultivars from Fragaria spp. showed that the dominant gene was not rare in the germ plasm of F. x ananassa and that anthracnose resistance was also present in other species of Fragaria. These findings have important implications for anthracnose resistance breeding.     

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.     

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.