Identification and genetic diversity of Mycosphaerella species on banana and plantain in Nigeria

Ribosomal coding DNA was sequenced and compared in 95 isolates of Mycosphaerella spp. collected in Nigeria and single nucleotide polymorphism (SNP) was used to identify the species and to determine the genetic structure of the sampled geographical populations. Using reference GenBank accessions with intercontinental distributions as controls, and shared species-specific SNPs in these control accessions, 84 (88·4%) isolates that grouped into 14 SNP haplotypes were identified as M. fijiensis , while 11 (11·6%) isolates represented by seven SNP haplotypes were characterized as M. eumusae . None of the isolates were either M. musicola or M. musae . The presence of M. fijiensis and M. eumusae in the collection was further confirmed using previously published species-specific probes designed on actin and β-tubulin gene sequences. A pairwise comparison of the population genetic distances revealed significant genetic differentiation between most populations ( P  < 0·001), with an average F _ST of 0·126, and a population structure corresponding to the four sampled geographical zones. The intraspecific dissimilarity of M. eumusae was 4·6%, compared with 2% for M. fijiensis . Compared to all the GenBank reference accessions, three sequence variations were unique to some Nigerian M. fijiensis haplotypes. Twenty-one sequence haplotypes were identified, geographically mapped and registered in GenBank. The results indicate that M. musicola has been replaced by more frequently occurring M. fijiensis and M. eumusae , against which disease management and resistance breeding efforts should be directed in Nigeria.     

Genetic structure of Mycosphaerella fijiensis populations from Australia, Papua New Guinea and the Pacific Islands

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Mycosphaerella fijiensis , the cause of black leaf streak (black Sigatoka) disease of banana and plantain, in the Torres Strait, Papua New Guinea (PNG), and the Pacific Islands. A moderate level of genetic variation was observed in all populations with genotypic diversity values of 60–78% of the theoretical maximum, and gene diversity ( H ) values between 0·269 and 0·336. All populations were at gametic equilibrium, and with the high level of genotypic diversity observed this indicated that sexual reproduction has a major role in the genetic structure of the M. fijiensis populations examined. Population differentiation was tested on several hierarchical scales. No evidence of population differentiation was observed between sites on Mer Island. A moderate level of population differentiation was observed within the Torres Strait, between Badu and Mer Islands ( F _ST = 0·097). On a regional scale, the greatest differentiation was found between the populations of the Torres Strait and the Pacific. Populations from these regions were more closely related to the PNG population than to each other, suggesting they were founded in separate events from the same population.     

Diversity and differentiation in two populations of Gibberella circinata in South Africa

Gibberella circinata [anamorph Fusarium circinatum (=  F. subglutinans f.sp. pini )] causes pitch canker and is an important pathogen in South African pine nurseries. The initial outbreak of the pitch canker fungus was limited to a single nursery at Ngodwana in Mpumalanga Province. Subsequently, several other pine nurseries in South Africa became infected. Most of these outbreaks were relatively small except for the outbreak in the Klipkraal nursery (Mpumalanga Province). The genetic diversity, population differentiation and relative frequencies of the sexual and asexual cycles among two South African subpopulations were determined to establish whether immigration, mutation and/or recombination contributed towards population structure. The allelic diversity of the initial population (Ngodwana) was observed to be lower (0·16) than that of the more recent Klipkraal population (0·25). Approximately 4% ( G _ST = 0·04) of total gene diversity could be attributed to differences among the subpopulations. Furthermore, six new vegetative compatibility groups (VCGs) have been identified since the initial outbreak of G. circinata in South Africa 10 years ago. The relatively low allelic diversity and low level of genetic differentiation suggest restricted gene flow among subpopulations, and indicate that the pathogen has been introduced recently. However, the amount of allelic and VCG diversity suggests that multiple genotypes have been introduced into South Africa. The increases in effective population number, allelic diversity and new VCGs over the past 10 years suggest that sexual reproduction might be occurring.     

Genetic diversity of Claviceps africana on sorghum and Hyparrhenia

RAPD markers were used to survey genetic variability among 140 isolates of Claviceps africana collected from Southern Africa, India, Thailand, Australia and the Americas in 1992–2002. Amplified fragment length polymorphisms were determined for a subset of the isolates. Both markers gave similar results in phenetic analysis of genetic distances between haplotypes of different geographical origin. In the Americas, a single RAPD haplotype was found throughout the various countries. The Eastern lineage consisted of two close haplotypes (one from India, the other from Thailand and Australia). Among five specialized isolates of C. africana from the alternative hosts ( Hyparrhenia spp.), three haplotypes were found. Eleven private alleles distinguished the Hyparrhenia population from that on sorghum. rDNA sequences of sorghum and Hyparrhenia isolates differed in three positions. The African sorghum population of C. africana consisted of 10, mostly closely related haplotypes. Low genotypic diversity ( H _E = 0·0337) and the fact that most of the variation originated from between populations ( G _ST = 0·866) suggested founder effects following recent invasion. In Southern Africa, no significant differentiation was found among six populations. Therefore the data were pooled and tested for prevalence of clonal or sexual reproduction. The presence of the over-represented, widespread RAPD haplotype A; gametic disequilibrium (37% loci detected by exact tests); index of association ( I _A) significantly >0; and the high proportion of compatible loci (in the clone-corrected and total data sets found to be 94 and 99%, respectively) support the hypothesis of clonality as the predominant means of reproduction.     

Population structure and mating system of Ascochyta rabiei in Tunisia: evidence for the recent introduction of mating type 2

The population structure of Ascochyta rabiei (teleomorph: Didymella rabiei ) in Tunisia was estimated among five populations sampled from the main chickpea growing regions using simple sequence repeat markers (SSR) and a mating type ( MAT ) marker. Mating type 2 isolates ( MAT1-2 ) had reduced genetic and genotypic diversity relative to mating type 1 isolates ( MAT1-1 ). This result, coupled with previous observations of lower overall frequency and restricted geographical distribution of MAT1-2 in Tunisia, and recent (2001) observation of the sexual stage, support the hypothesis of a recent introduction of MAT1-2 . Despite the presence of both mating types in Nabeul, Kef and Jendouba, the hypothesis of random mating was rejected in these locations with multilocus gametic disequilibrium tests. Highly significant genetic differentiation ( θ  = 0·32, G _ST = 0·28, P  < 0·001) was detected among populations and genetic distance and cluster analyses based on pooled allele frequencies revealed that populations from Nabeul and Kef were distinct from those in Beja, Bizerte and Jendouba. More than 70% of total gene diversity ( H _T = 0·55) detected was attributable to variation within populations compared to 28% among populations. This result, coupled with the occurrence of private alleles in each population, suggests that gene flow is currently limited among populations, even those separated by short geographic distances. The presence of two main genetic clusters was confirmed using Bayesian model-based population structure analyses of multilocus genotypes (MLGs) without regard to geographic origin of samples. The presence of MAT1-2 isolates in both clusters suggests at least two independent introductions of MAT1-2 into Tunisia that are likely to be the result of importation and planting of infected chickpea seeds.     

Genetic variation among field isolates of Pyrenopeziza brassicae

Amplified fragment length polymorphism (AFLP) markers were used to determine genetic diversity and population structure of Pyrenopeziza brassicae , the causal agent of light leaf spot of Brassica spp. Fungal isolates were sampled from six regions in the UK, one region in Germany and one region in France. A high level of genetic diversity was found ( H _T = 58%), with most variation attributed to within regions ( H _S = 43%), which suggests that sexual reproduction is frequent. F _ST values suggested significant population differentiation between England and the continent, but not between Scotland and England and Scotland and the continent. Overall, a moderate but significant level of regional differentiation was found ( F _ST = 16 ± 4.0). There was no correlation between F _ST values and distance, indicating that long-distance dispersal by natural factors does not occur at high frequencies. However, the lack of differentiation among populations from Aberdeen, Winchester and Cambridge suggests that seed transmission or other artificial methods of dispersal may be important.     

Genetic variation among populations of Pythium irregulare in southern Australia

Isolates of Pythium irregulare were sampled from seven cereal crops throughout South Australia to determine the extent of genetic diversity within this pathogen and the scale of genetic differentiation among populations. Data derived from 29 individual restriction fragment length polymorphism (RFLP) loci differentiated 54 DNA fingerprints among the 92 isolates analysed. Some isolates had two alleles at several RFLP loci and were scored as heterozygous. One such isolate was selfed in vitro and segregation ratios in the progeny were not significantly different from those expected for allelic variation in a diploid. These data provided evidence that outcrossing occurs within P. irregulare and may contribute to the high level of genetic variation within the species ( D _T = 0·502). Allelic frequencies were significantly different among all seven populations and G _ST values showed significant genetic differentiation between populations. The average genetic identity among populations was low and hierarchical cluster analysis provided no clear evidence that populations formed geographically related groups. These analyses indicate low levels of interpopulation gene flow within P. irregulare and imply that population differentiation results from genetic drift.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

The Genetic Structure of Australian Populations of Mycosphaerella musicola Suggests Restricted Gene Flow at the Continental Scale

ABSTRACT Mycosphaerella musicola causes Sigatoka disease of banana and is endemic to Australia. The population genetic structure of M. musicola in Australia was examined by applying single-copy restriction fragment length polymorphism probes to hierarchically sampled populations collected along the Australian east coast. The 363 isolates studied were from 16 plantations at 12 sites in four different regions, and comprised 11 populations. These populations displayed moderate levels of gene diversity (H = 0.142 to 0.369) and similar levels of genotypic richness and evenness. Populations were dominated by unique genotypes, but isolates sharing the same genotype (putative clones) were detected. Genotype distribution was highly localized within each population, and the majority of putative clones were detected for isolates sampled from different sporodochia in the same lesion or different lesions on a plant. Multilocus gametic disequilibrium tests provided further evidence of a degree of clonality within the populations at the plant scale. A complex pattern of population differentiation was detected for M. musicola in Australia. Populations sampled from plantations outside the two major production areas were genetically very different to all other populations. Differentiation was much lower between populations of the two major production areas, despite their geographic separation of over 1,000 km. These results suggest low gene flow at the continental scale due to limited spore dispersal and the movement of infected plant material.     

应用RAPD标记技术鉴定香蕉褐缘灰斑病菌

采用Mycosphaerella fijiiensis和M.musicola的RAPD标记技术鉴定海南香蕉褐缘灰斑病菌,结果表明,分离自海南儋州、乐东、文昌、东方、澄迈、临高、琼海、昌江、琼山、三亚香蕉上的10个菌株均为M.fijiensis,引起香蕉黑叶条斑病。     

Genetic variation in Danish populations of Erysiphe graminis f.sp. hordei: estimation of gene diversity and effective population size using RFLP data

Genetic variation of the barley powdery mildew fungus ( Erysiphe graminis f.sp. hordei ) was estimated in three Danish local populations. Genetic variation was estimated from the variation amongst clones of Egh , and was therefore an estimate of the maximum genetic variation in the local populations. The average gene diversity, Ĥ , was estimated as 0.84. The effective population size was estimated as: log₁₀ ( N^ _e ) = 0.64 − log₁₀(μ), or 4.4 × 10⁹, assuming a nucleotide mutation rate ( μ) of 10⁻⁹ per base per generation. There was no significant genetic differentiation between locations.     

Host–parasite relationships of Meloidogyne incognita on spinach

Host–parasite relationships in root-knot disease of spinach caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Nematode-induced mature galls were large and usually contained one or more females and egg masses with eggs. Feeding sites were characterized by the development of giant cells containing granular cytoplasm and many hypertrophied nuclei. The cytoplasm in these giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P _i) in a series from 0–128 eggs and second-stage juveniles per cm³ soil and growth of spinach cv. Symphony F₁ seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^P–T ] was fitted to fresh top- and total plant-weight data for inoculated and control plants. Tolerance limits ( T ) of spinach cv. Symphony F₁ to M. incognita race 1 for fresh top and total plant weights were 0·25 and 0·5 eggs and second-stage juveniles per cm³ soil, respectively. The minimum relative values for fresh top and total plant weights were zero in both cases at P _i ≥ 32 eggs and second-stage juveniles per cm³ soil. Root galling was least at low initial population densities and greatest at 16 eggs and second-stage juveniles per cm³ soil. Maximum nematode reproduction rate was 33·1-fold at the lowest P _i.     

Effect of water potential on mycelial growth and perithecial production of Monosporascus cannonballus in vitro

The effects of osmotic water potential (Ψ_s) on mycelial growth and perithecial production of Monosporascus cannonballus , the cause of root rot and vine decline of melons, were examined at 25°C on potato dextrose agar (PDA) amended with KCl, NaCl or sucrose. Patterns of the growth responses of four isolates to decreasing Ψ_s were similar for each of the osmotica. Compared with growth on nonamended PDA (−0·3 MPa), growth of all isolates increased as Ψ_s was reduced to −0·8 MPa. Maximum growth occurred at Ψ_s values of −0·6 to −0·8 MPa. Growth was not reduced below that on nonamended PDA until Ψ_s was reduced to −1·8 MPa, and a 50% reduction in growth did not occur until Ψ_s was reduced to < −2·5 MPa. Reproduction was much more sensitive to reduced Ψ_s than was mycelial growth, and perithecia were produced only at Ψ_s ≥ −0·7 or −0·8 MPa on PDA amended with KCl or NaCl, respectively. Three isolates produced perithecia on PDA amended with sucrose only at Ψ_s ≥ −0·6 MPa, but the fourth isolate produced perithecia at ≥ −1·9 MPa. Colonization of the xylem early in disease development may provide an essential source of water for subsequent reproduction in the root cortex during plant senescence. Postharvest cultivation to expose and desiccate roots may prevent reproduction even when temperatures lethal to hyphae are not attained.     

Population structure of poplar rust Melampsora larici-populina in the UK inferred from AFLP

AFLP profiles were generated from poplar leaf rust ( Melampsora larici-populina ) collected from Populus trichocarpa cv. Trichobel at six sites in the UK in 2001. Of a total of 145 isolates, 121 were unique AFLP phenotypes. On one occasion, the same AFLP phenotype was found at two sites. The analysis indicated that 81·88% of the total variation was attributable to differences among individuals within replicate plots, 3·47% to differences among plots within sites and 14·65% to differences among sites. Weir & Cockerham's θ was calculated as 0·138 and θ ^B , the Bayesian analogue of F _ST, as 0·146. Overall, there was sufficient gene flow among different sites to prevent genetic drift, as Nm was calculated as 1·562 using θ and as 1·462 using θ ^B . Nei's genetic distance and population-assignment test indicated that the rust from a site in north-east England was differentiated from those from other sites. There was no significant correlation between genetic and geographical distances. The results indicate that sexual reproduction has a major influence on the relative lack of population structure of M. larici-populina in the UK.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Variation among Colletotrichum gloeosporioides isolates from infected coffee berries at different locations in Vietnam

Isolates of Colletotrichum gloeosporioides associated with anthracnose disease on coffee berries in Vietnam were characterized by morphological and molecular methods. Random amplified polymorphic DNA (RAPD) and microsatellite-primered PCR (MP-PCR) analyses were employed to investigate the genetic variation among 38 and 51 isolates of C. gloeosporioides , respectively. According to both methods, the isolates mainly grouped in accordance with geographical origins. Higher genetic variation ( H  = 0·312 and 0·335) in the northern population of C. gloeosporioides than in the southern population ( H  = 0·261 and 0·186), according to the RAPD and MP-PCR markers, respectively, was indicative of a difference between the northern and southern populations. Moderate gene differentiation ( G st = 0·1) between populations from the north and the south was found. However, there was no differentiation between locations within the northern or southern populations, indicating significant gene flow. A four-gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam. The symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD (coffee berry disease; Colletotrichum kahawae ) isolates originating from Africa.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Genetic diversity of Australian Fusarium graminearum and F. pseudograminearum

Genotypic diversity in Fusarium pseudograminearum and F. graminearum from Australia and the relationship between diversity and pathogen aggressiveness for head blight and/or crown rot of wheat were examined. Amplified fragment length polymorphism (AFLP) analysis revealed a high level of genotypic diversity within each species. Sixty-three of the 149 AFLP loci were significantly different between the two species and 70 of 72 F. pseudograminearum and 56 of 59 F. graminearum isolates had distinct haplotypes. When head blight and crown rot severity data from a recently published work on isolates representing the entire range of aggressiveness were used, only the genotypic diversity of F. pseudograminearum was significantly associated with its aggressiveness for the two diseases. Cluster analyses clearly demonstrated the polyphyletic structures that exist in both pathogen populations. The spatial diversity within F. graminearum was high within a single field, while frequent gene flow ( N _m ∼ 14) and a low fixation index ( G _st = 0·03) were recorded among F. pseudograminearum isolates from the adjacent states of New South Wales and Queensland. The differences in population structure between the heterothallic F. pseudograminearum (teleomorph G. coronicola ) and the homothallic F. graminearum (teleomorph G. zeae ) were not as pronounced as expected given their contrasting mating systems. Neither species was panmictic or strictly clonal. This points to sexual recombination in F. pseudograminearum , suggesting that ascospores of G. coronicola may also play a role in its biology and epidemiology.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

Control of Penicillium expansum by plant volatile compounds

Nine plant volatiles were tested for their activity in vitro and in vivo against Penicillium expansum , the cause of blue mould of pear. In vitro spore germination and mycelial growth assay showed a consistent fungicidal activity by trans -2-hexenal, carvacrol, trans -cinnamaldehyde and citral, while hexanal (-)- carvone, p -anisaldehyde, eugenol and 2-nonanone exhibited a progressively lower inhibition. trans -2-Hexenal was the best inhibitor of conidial germination [MIC (minimum inhibitory concentration) = 24·6  µ L L⁻¹; ED₅₀ = 10·2  µ L L⁻¹], while carvacrol was the best inhibitor of mycelial growth (MIC = 24·6  µ L L⁻¹; ED₅₀ = 9  µ L L⁻¹). The four most active compounds in in vitro studies were tested in vivo as fumigants against blue mould on pear cv. Conference. Best control was achieved by trans -2-hexenal vapour treatments (12·5  µ L L⁻¹) when applied over a 24-h period, beginning 24 h after inoculation. In contrast, carvacrol (12·5–200  µ L L⁻¹), and trans -cinnamaldehyde (50–400  µ L L⁻¹) were ineffective and citral (200  µ L L⁻¹) showed only slight effect.