Averil E. Brown

DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose

Abstract Restriction fragment length polymorphisms (RFLPs) of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the strawberry anthracnose pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. acutatum, C. fragariae and C. gloeosporioides as probes. These analyses revealed considerable heterogeneity within C. acutatum from diverse hosts. The European strawberry isolates formed a discrete group while the American strawberry isolates fell into a broad group which included isolates from other hosts. No polymorphisms in either rDNA or mtDNA were observed among C. gloeosporioides isolates from strawberry. C. fragariae isolates divided into two groups with distinct rDNA and mtDNA patterns. Random amplified polymorphic DNA (RAPD) analysis grouped isolates in a similar manner to the mtDNA RFLP analysis. From sequencing data, the internally transcribed spacer (ITS) 1 region of the rDNA repeat unit of C. gloeosporioides, C. fragariae and C. acutatum was shown to be 171, 171 or 172 and 180 or 181 bases, respectively. C. gloeosporioides and C. fragariae differed from one another by only three to seven bases compared with C. acutatum which differed from C. gloeosporioides and C. fragariae by approx. 36–37 bases. ITS 1 data for C. acutatum generally support RFLP and RAPD taxonomies; differences of eight-eleven bases between European strawberry isolates and all other isolates studied being the greatest.

Nucleotide sequence of the rDNA spacer 1 enables identification of isolates of Colletotrichum as C. acutatum.

Nucleotide sequence of the rDNA internally transcribed spacer (ITS) 1 of twelve isolates of Colletotrichum , originally identified as either C. fructigenum, C. gloeosporioides or C. musae , was determined. Comparison of the sequence data with that from previously characterized isolates of Colletotrichum species revealed 97–100% homology with C. acutatum and only 79–81% homology with C. gloeosporioides and C. musae . We conclude that the isolates belong to C. acutatum . The value of sequence data from ITS 1 in species assignment is demonstrated.

Cross-infection potential of genetic groups of Colletotrichum gloeosporioides on tropical fruits

Cross-inoculation experiments demonstrated variation in the level of host preference among Colletotrichum gloeosporioides isolates from seven tropical fruit crops and also variation in the susceptibility of the hosts. In general, isolates were much more pathogenic on leaves of the original host than on those of alternative crops. Isolates from avocado, durian and mango showed the greatest degree of host preference while isolates from mangosteen and pini jambu were the most pathogenic on alternative crops. Avocado, mango and rambutan were the most susceptible and mangosteen and pini jambu were the least susceptible to C. gloeosporioides isolates from the other crops. The extent of cross-infection appeared dependent on inoculum density. Molecular markers based on restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA), mitochondrial DNA (mtDNA) and random amplified polymorphic DNA (RAPD) enabled genetic grouping of the C. gloeosporioides isolates. In some instances, isolates from different hosts, e.g. two of the avocado isolates, all three durian isolates and one rambutan isolate, had identical rDNA and very similar mtDNA banding patterns (category 3) indicating a common ancestry, but differed in their host preference, implying adaptation to different crops.

Coffee berry disease pathogen in Africa: genetic structure and relationship to the group species Colletotrichum gloeosporioides

Restriction fragment length polymorphisms of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the coffee berry disease pathogen in Africa, Colletotrichum kahawae , were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. kahawae and C. gloeosporioides as probes. These analyses revealed homogeneity among C. kahawae isolates. The estimated sizes of the rDNA repeat unit and the mtDNA of C. kahawae were 8·4–9·18 kb and 60 kb, respectively. Polymorphisms were observed in rDNA and mtDNA when C. kahawae was compared with C. gloeosporioides from coffee, avocado and mango. However, an avocado isolate JIA1, from Australia, had an identical rDNA restriction pattern to C. kahawae when digested with Bam H I and C. kahawae showed greater than 96% similarity to two C. gloeosporioides avocado isolates (918 and 1072) from New Zealand in mtDNA restriction fragment pattern. Random amplified polymorphic DNA analysis also grouped the C. kahawae isolates together. Nucleotide sequence of the internally transcribed spacer 1 region of the rDNA repeat unit of C. kahawae and C. gloeosporioides isolates from Coffea spp. differed by only two to three bases (98·8–98·2 % homology). Results obtained confirmed the close genetic relationship of C. kahawae to the group species C. gloeosporioides .

Antifungal compounds produced by Epicoccum purpurascens against soil-borne plant pathogenic fungi

Abstract Epicoccum purpurascens inhibited colony growth of Phytophthora spp and Pythium spp in culture more than other species tested. Six antifungal compounds were detected in cultures of E. purpurascens grown in two selective media. Four of these compounds, epicorazines A and B and two compounds (X and Y) of unknown identity (designated the epicorarine fraction) were produced simultaneously at an early stage in the growth of E. purpurascens in a sucrose plus casamino acid medium but were not detected in a glucose plus (NH4HPO4 medium. The compound flavipin was detected in both media but was preferentially produced in the latter. A third unidentified antifungal compound was detected in both media at a late growth stage. Mycelial growth of Pythium spp was more sensitive to the epicorazine fraction than to flavipin. Phytophthora spp tended to be more sensitive to flavipin. Both were much less toxic to mycelial growth of Fusarium oxysporum f.sp. lini. Flavipin inhibited germination of zoospores of Phytophthora cinnamomi and oogonia of Pythium intermedium. Mycelia of Phytophthora spp and Pythium spp antagonized by E. purpurascens were stunted and swollen and where opposing cultures intermingled hyphae of E. purpurascens coiled and penetrated those of the two pathogens. Cellulase and β, 1–3 glucanase activities were detected in cultures of E. purpurascens grown on Phytophthora or Pythium spp hyphal wall material as the sole carbon source.

Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method

Cladobotryum dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the β-tubulin gene were used to amplify and sequence a portion of the β-tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the β-tubulin gene containing this substitution. The point mutation removed an Acc I restriction site in the β-tubulin gene sequence of resistant isolates. Digestion of the PCR product with Acc I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: Y12256.

Ribosomal and mitochondrial DNA polymorphisms in Colletotrichum gloeosporioides isolated from tropical fruits

DNA polymorphisms in 38 isolates of Colletotrichum gloeosporioides infecting avocado, banana, mango and papaya were examined using ribosomal DNA (rDNA) from Saccharomyces carlsbergensis and mitochondrial DNA (mtDNA) purified from C. gloeosporioides total DNA as probes. The average size of the rDNA repeat unit of the isolates was 9·2 kb, although there was considerable size variation. Isolates obtained from different hosts never had the same rDNA or mtDNA RFLP patterns. With the exception of those from mango, isolates could not easily be grouped but could be distinguished in relation to their host source within geographical localities. There was no RFLP that could be used diagnostically on a worldwide basis to identify isolates from avocado, banana or papaya. In contrast, isolates obtained from mango fruits in the eastern and western hemisphere had the same rDNA and very similar mtDNA restriction fragment-banding patterns.

Genetic variation among and within Monilinia species causing brown rot of stone and pome fruits

Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) divided the three brown rot pathogens Monilinia laxa, M. fructicola and M. fructigena into four distinct groups. Isolates of M. fructigena received from Japan, which varied by 5 base substitutions in the ITS region from the European M. fructigena isolates, formed the fourth group. Four of five Japanese isolates of M. fructicola tested varied from the New World isolates in that they did not possess a group-I intron in the small subunit (SSU) rDNA. RAPD-PCR data indicated that isolates of M. laxa varied but were randomly distributed worldwide; ITS data indicated no apparent distinction between those from Malus spp. and those from Prunus spp. M. fructigena similarly did not cluster according to geographic origin. In contrast, M. fructicola isolates tended to be clustered according to their origin; Japanese isolates of M. fructicola clustered together and showed similarity to some of the New Zealand isolates. Isolates from USA and Australia were more variable.

Production of antibiotics by Bacillus subtilis and their effect on fungal colonists of apple leaf scars

Apple rootstocks were sprayed after leaf fall with two antibiotic-yielding isolates of Bacillus subtilis. Both could be recovered from the leaf scar tissue throughout the dormant season and spring, but with time an increasing number of strains of B. subtilis were isolated, all of which possessed both antifungal and antibacterial activity similar to that of the original strains used. B. subtilis did not persist on the rootstock bark. The antifungal metabolites produced by B. subtilis were shown to be relatively stable compounds in vitro but the antibacterial metabolites were rapidly inactivated. The antifungal metabolites were found to be as inhibitory to many of the fungi commonly isolated from apple leaf scar tissue as they were to Nectria galligena.

Phenotypic and genotypic characterisation of Northern Ireland isolates of Phytophthora infestans

Genetic and phenotypic diversity of the population of the late blight pathogen Phytophthora infestans in Northern Ireland was assessed from 223 single-lesion isolates collected from 29 sites in 1995 and 1996. The proportion of metalaxyl-resistant isolates was approximately 14%. The A2 mating type was not detected. Allozyme analyses revealed that the Northern Ireland isolates were monomorphic and homozygous at the loci coding for allozymes of glucose-6-phosphate isomerase and peptidase (Gpi 100/100, Pep 100/100). The majority of isolates tested (156 out of 162) were mitochondrial DNA haplotype IIa. Among the remaining isolates, four were haplotype Ia, two were the rare IIb haplotype and none was of the old Ib type. RAPD-PCR analysis of selected isolates revealed relatively little diversity among the Northern Ireland isolates, which clustered separately from isolates from GB and the European mainland.