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.     

<Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> occurs asymptomatically on apple leaves

Fungi within the Colletotrichum acutatum species complex occur asymptomatically on plant parts of many different plant species. Leaves from apple orchards in southern Norway were sampled, frozen for five hours and incubated for six days to reveal presence of asymptomatic infections of C. acutatum. Number of leaves (incidence) and leaf area covered (severity) with conidial masses of C. acutatum were assessed biweekly on cv. Aroma from late May to late September during three growing seasons. The first finding of conidial masses occurred in the second half of July, and there was a higher incidence occurring in August and September. Sampling of leaves from fruit spurs and vegetative shoots of cvs. Aroma and Elstar showed that conidial masses of C. acutatum developed on leaves on both shoot types, and there was no difference in incidence between these two types. The fungus was detected on leaves from six of eight commercial orchards of cv. Aroma over three years, with a mean incidence of 5.5 %. After storage, bitter rot was found on apple fruit from all eight orchards. There was no correlation between incidence of conidial masses of C. acutatum on leaves and on fruit. In all orchards and seasons investigated, incidence and severity on leaves varied from 0 to 67 % and 0 to 85 %, respectively. The discovery of apple leaves containing conidial masses of C. acutatum clearly indicate for leaves as a potential source of inoculum for fruit infections.     

A rapid qualitative molecular method for the identification of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> and <Emphasis Type="Italic">C. gloeosporioides</Emphasis>

Identification of the causal agent for anthracnose caused by C. acutatum and C. gloeosporioides based on morphological and cultural criteria is problematic as both are morphologically and genetically diverse. To evaluate a qualitative molecular method to readily distinguish between these two species, Restriction fragment length polymorphisms (RFLP) of a 1-kb intron of the glutamine synthetase (GS) gene was evaluated utilizing representative isolates from a world-wide collection. Unique band patterns of the 1-kb GS intron were obtained for C. acutatum (two fragments with 600 and 350 bp) and C. gloeosporioides (four fragments with 238–340, 252–254, 204, and 108–116 bp) based on PstI enzyme digestion of the amplified PCR product. These data were also confirmed by PstI digestion of the intron DNA sequences using BioEdit software. The identification based on RFLPs of the 1-kb GS intron was consistent with the identification based on previously evaluated species-specific primers (CaInt2 and CgInt). In addition, both species can be differentiated by multiplex PCR. CaInt2, CgInt and ITS4 in one PCR will distinguish between C. acutatum and C. gloeosporioides by differences in PCR product fragment size: 490 bp and 470 bp, respectively. Also, a rapid DNA extraction method was developed, which reduced the time for DNA extraction from two hours to five minutes. In summary, RFLP of the 1-kb GS intron is a reliable technique for identification and differentiation between both species, does not require a sequencing step, and may be useful to diagnostic clinics in helping to make disease management recommendations.     

Pathogenicity and characterization of <Emphasis Type="Italic">Colletotrichum lentis</Emphasis>: A causal agent of anthracnose in common vetch (<Emphasis Type="Italic">Vicia sativa</Emphasis>)

A series of studies were carried out on Colletotrichum lentis which had been been identified in 2015 based largely on the distinctive shape of conidia and ITS sequences, and which has been causing severe anthracnose disease symptoms on common vetch plants (Vicia sativa) in Gansu Province in the northwest region of China. A key focus of the present studies was to determine how vetch crops become infected. The addition of residues from harvested common vetch crops to land being prepared as a seedbed was shown to result in the highest levels of disease severity indicating that this management practice was the most likely way for crops to become severely infected. Seed transmission was unlikely to be the cause of severe outbreaks as less than 5% of seeds harvested from severely infected plants carried C. lentis. To verify that the species causing the severe outbreaks of anthracnose disease of vetch crops was C. lentis, sequence analysis of the ITS, TUB2, ACT, HIS3 and GAPDH genes was conducted. C. lentis isolates from common vetch and lentil (Lens culinaris) formed a distinctive group among Colletotrichum species, including those species that infect other forage and field crops. The unique shape of conidia of C. lentis, straight with only one end curved, was confirmed as being reliable for rapid identification of disease outbreaks caused by this damaging fungal pathogen.     

<Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> occurs asymptomatically on sweet cherry leaves

Leaves of sweet cherry, exposed to either paraquat or freezing to quickly senesce the leaf tissue, were incubated in about 100% RH at 25°C for 6 d. Sporulating colonies of Colletotrichum acutatum, the cause of anthracnose, developed on up to 100% of the paraquat-treated and frozen leaves, and on none of the untreated controls. Number of leaves and leaf area containing C. acutatum on naturally infected leaves increased over time from May to September. Mean incidence of C. acutatum on leaf blades on fruit spurs and vegetative shoots from eight orchard/year samplings were 41 and 33%, respectively. Secondary conidiation (formation of short hyphae and new conidia) from conidia applied to detached leaves took place 6 h after inoculation, but only up to 3% of the conidia formed new conidia. It may be concluded that asymptomatic sweet cherry leaves frequently host C. acutatum and may be a potential inoculum source for cherry fruit.     

Anthracnose of three-leaf akebia (<Emphasis Type="Italic">Akebia trifoliata</Emphasis> Koidzumi) caused by <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

In October 2001, anthracnose caused by Colletotrichum acutatum Simmonds ex Simmonds was found on three-leaf akebia (Akebia trifoliata) in Saitama, Japan. This is the first report of anthracnose on three-leaf akebia caused by C. acutatum.     

Markers to differentiate species of anthracnose fungi identify <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> as the predominant virulent species in strawberry plants in Chiba Prefecture of Japan

Colletotrichum fungi belonging to the Colletotrichum gloeosporioides species complex include a number of economically important postharvest pathogens that often cause anthracnose. Until now, different species within this group could only be distinguished from one another reliably using multigenic phylogenetic analyses. Using a comparative genomics approach, we developed a marker that can differentiate Colletotrichum fructicola, Colletotrichum aenigma and Colletotrichum siamense within the C. gloeosporioides species complex based on PCR amplicon size differences. When we used this marker to classify 115 isolates collected over 20 years from strawberries in Chiba Prefecture, Japan, the isolates were predominantly C. fructicola. To our knowledge, this is the first report characterizing different species of Colletotrichum infecting strawberries in Japan and contributes to our understanding on the diversity of anthracnose pathogens in Japan.     

Anthracnose of <Emphasis Type="Italic">Polygonatum falcatum</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum dematium</Emphasis>

Severe spotting, blight and drop of leaves caused by Colletotrichum dematium were found on potted plants of Polygonatum falcatum, a liliaceous ornamental, in open fields in Kagawa Prefecture, Japan, in May 2001. This new disease was named anthracnose of P. falcatum. Keisuke Tomioka, Jouji Moriwaki, Toyozo Sato contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under accessions MAFF239500 and AB334523, respectively.     

Postharvest anthracnose on Satsuma mandarin orange caused by <Emphasis Type="Italic">Colletotrichum fioriniae</Emphasis>

In early January 2015–2017, anthracnose was detected on Satsuma mandarin orange (SMO) (Citrus unshiu) fruits kept in farmers’ storage rooms in Saga Prefecture, Japan. Three single-spore isolates from rotten fruits reproduced the postharvest anthracnose symptoms in wound-inoculated SMO fruits and were re-isolated from lesions. The isolates were identified as Colletotrichum fioriniae based on conidial morphology, culture characteristics, rDNA-ITS, and beta-tubulin-2 gene sequences. This is the first report of postharvest anthracnose on SMO caused by C. fioriniae.     

Assessment of the baseline sensitivity and resistance risk of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> to fludioxonil

Anthracnose, which is caused by Colletotrichum acutatum, is a destructive disease of pepper. A preliminary study demonstrated that fludioxonil (a phenylpyrrole fungicide) has good activity against C. acutatum and thus has potential to be used as an alternative fungicide for the management of pepper anthracnose. However, there is no information regarding the baseline sensitivity and resistance risk of C. acutatum to fludioxonil. Thus, the sensitivities of 205 isolates of C. acutatum to fludioxonil were determined. The results showed that the frequency distributions of the EC₅₀ values were unimodal, and the mean EC₅₀ values for the inhibition of mycelial growth and spore germination were 0.031 μg/mL and 0.035 μg/mL, respectively. Three stable mutants with high resistance to fludioxonil were obtained in the laboratory. Two parameters, namely in vitro sporulation and the in vitro and in vivo germination of spores, showed significant difference (P < 0.01) when the mutants were compared to the sensitive isolates. Moreover, the mutants were more sensitive to osmotic stress compared to the parents. No significant differences (P ≥ 0.05) were detected in colony diameter, mycelia weight, pathogenicity or sporulation in vivo between the fludioxonil-resistant mutants and their corresponding parents. Cross-resistance occurred between fludioxonil, iprodione and procymidone. Overall, resistance risk of C. acutatum to fludioxonil was low to medium, and thus resistance management should be considered.     

Weeds as the potential inoculum source of <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> responsible for strawberry anthracnose in Nara,Japan

Colletotrichum fructicola is a major causal agent among anthracnose pathogens of strawberry in Nara, Japan. We hypothesized that a wide range of weeds growing in and around strawberry fields are inoculum sources of the disease and investigated their potential as hosts of C. fructicola. We also examined the influence of herbicide treatment on C. fructicola sporulation on weeds. The fungus was detected on 31 of 541 (5.7%) leaves sampled from 13 weed species from 2005 to 2008. The fungus was most frequently isolated from leaves of Amaranthus blitum with an isolation frequency of 17.9%; inoculation of A. blitum with the pathogen caused brown leaf spots. Other weeds such as Digitaria ciliaris, Galinsoga ciliata, Solidago altissima, Erigeron annuus, and Sonchus oleraceus were found to harbor the fungus at lower rates (4.3–8.1%) without symptoms. C. fructicola formed acervuli on leaves of A. blitum, D. ciliaris, and S. oleraceus after plants were killed by a herbicide (glyphosate). These results demonstrated that infected weeds associated with strawberry cultivation are potential inoculum sources of C. fructicola, especially after herbicide treatment.     

Anthracnose of <Emphasis Type="Italic">Enkianthus campanulatus</Emphasis> and <Emphasis Type="Italic">Rhynchosia acuminatifolia</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> (new occurrence)

In 2003–2004, anthracnoses of Enkianthus campanulatus and Rhynchosia acuminatifolia were found for the first time in Kanagawa Prefecture and Tokyo in Japan. These pathogens were identified as Colletotrichum gloeosporioides based on their pathogenicity, morphology and ribosomal DNA spacer sequences. Results were presented at the annual meeting of The Phytopathological Society of Japan in 2004.     

<Emphasis Type="Italic">Colletotrichum destructivum from</Emphasis> cowpea infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and its identity to <Emphasis Type="Italic">C. higginsianum</Emphasis>

Colletotrichum isolates isolated from cowpea in the Hangzhou area of China were identified as C. destructivum based on morphological characteristics, pathogenicity tests, sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal DNA and the infection process. The ability of the C. destructivum isolates to infect Arabidopsis thaliana was investigated under laboratory conditions and showed a two-phase hemibiotrophic infection process. In addition, the sequences of the rDNA ITS region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. This article presents new evidence in support of C. higginsianum as a synonym of C. destructivum.     

The Eurotiomycete <Emphasis Type="Italic">Apinisia graminicola</Emphasis> as the causal agent of a leaf spot disease on the energy crop <Emphasis Type="Italic">Miscanthus</Emphasis> x <Emphasis Type="Italic">giganteus</Emphasis> in Northern Germany

Miscanthus x giganteus is a fast growing, perennial energy crop for temperate climates. Because of its high annual biomass production rates and its characteristics as a low-input crop, an expansion of field cultivation can be anticipated to cover increasing demands for sustainable biomass production. However, knowledge about pathogens that could have an impact on biomass production is still limited for M. giganteus. Here, we report about the isolation of the filamentous fungus Apinisia graminicola from necrotic leaf lesions of M. giganteus grown on a field trial plot in Northern Germany. Inoculation assays with the isolated A. graminicola strain confirmed its capacity to cause a leaf spot disease on M. giganteus. Additional inoculation assays revealed that A. graminicola also caused necrotic lesions on leaves of the model grass Brachypodium distachyon. Generally, symptoms of A. graminicola-caused leaf spot disease were stronger on B. distachyon compared to M. giganteus. Incubation temperatures above 22 °C during A. graminicola infection resulted in stronger disease symptoms on both, M. giganteus and B. distachyon leaves. Microscopic analysis of cross sectioned, infected leaf tissue revealed an epiphytic mycelium formation on the surface and an endophytic colonization of the mesophyll leave tissue, especially in M. giganteus. Our results revealed that the isolated A. graminicola strain is a causal agent of a leaf spot disease on grass leaves. Its potential on endophytic growth in M. giganteus might open new possibilities in studying this type of plant-fungal interaction on a cellular and molecular level in an energy crop.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

<Emphasis Type="Italic">Eutypa lata</Emphasis>, the causal agent of dieback in red currant (<Emphasis Type="Italic">Ribes rubrum</Emphasis>) and gooseberry (<Emphasis Type="Italic">R. uva-crispa</Emphasis>) in the Netherlands

Dieback of red currant (Ribes rubrum) and gooseberry (Ribes uva-crispa) is an increasing problem in commercial fields in the Netherlands. Field surveys were done in 2006–2007 and samples with dieback symptoms were analysed. In this study the causal agent was diagnosed as Eutypa lata, based on morphological characteristics and rDNA-ITS sequence data. The field surveys revealed the presence of the anamorph and teleomorph states of the fungus produced on dead infected currant wood. Eutypa lata is a vascular pathogen of many woody plants. Related fungi from the same family Diatrypaceae are difficult to distinguish from E. lata based on morphological features. The genetic variability of E. lata was compared by rDNA-ITS sequencing of isolates from different hosts and origins. Within the E. lata isolates little variability in the ITS sequences was observed. Phylogenetic analysis showed no clear subdivisions within the species. Eutypa lata strains isolated from the different hosts were closely related, indicating that there is no direct evidence for host specificity.     

Anthracnose of salt-wort (<Emphasis Type="Italic">Salsola komarovii</Emphasis>) caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis>

In July 2006, black rot was observed on the leaves of 4-leaf-stage seedlings of salt-wort (Salsola komarovii) in Yamagata Prefecture, Japan. We isolated two single-conidial isolates from the diseased leaves. Although colony appearance of the isolates was different from that of each other, both isolates were identified as Colletotrichum truncatum by morphology and molecular similarity. After inoculation of healthy salt-wort plants with the isolates, the isolates were reisolated from symptomatic plants. We thus propose a new disease, anthracnose of salt-wort.     

Alternative hosts of <Emphasis Type="Italic">Curtobacterium flaccumfaciens</Emphasis> pv. <Emphasis Type="Italic">flaccumfaciens</Emphasis>, causal agent of bean bacterial wilt

Alternative hosts are an important way of phytopathogenic bacteria survival between crop seasons, constituting a source of inoculum for the following crops. Bacterial wilt, caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), is one of the most important diseases for common bean, and little information is available about the host range of the bacterium. In this study, we assessed possible alternative hosts for Cff, especially those cultivated during winter, in rotation systems with common bean. Plants of barley, black oat, canola, radish, ryegrass, wheat and white oat, were assessed under field and greenhouse conditions. Cff colonized epiphytically all plant species and endophytically black oat, ryegrass, wheat and white oat plants assessed in the greenhouse assays. Under field conditions, Cff colonized all plant species by except radish. All bacterial strains re-isolated from the plants were pathogenic to common bean and identified as Cff by PCR with specific primers. Based on our results, the cultivation of bean crop in succession with barley, black oat, canola, ryegrass, wheat and white oat should not be recommended, mainly in areas with a history of bacterial wilt occurrence. In these cases, the better option for crop rotation during the winter is radish, a non-alternative host for Cff.     

<Emphasis Type="Italic">Mycosphaerella</Emphasis> species occurring on <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> in Portugal

Mycosphaerella leaf disease (MLD) is caused by species of Mycosphaerella and several anamorphic form genera that have been connected to Mycosphaerella. Until recently, MLD of eucalypts was largely ignored in Portugal. However, serious damage to Eucalyptus globulus has been reported since 1999 when frequent and severe defoliation of young trees was observed. The severity of this disease prompted a preliminary study of the Mycosphaerella species associated with major symptoms of a leaf blotch disease in commercial plantations of E. globulus in Portugal, which is presented here. The species were identified by molecular methods based on the ITS1-5.8S-ITS2 cluster, together with morphological characters. In addition to confirming the species previously recorded, Mycosphaerella vespa is reported for the first time from Portugal, while the status of Mycosphaerella grandis remains to be resolved.