High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.     

Genetic population structure and fungicide resistance of Botrytis cinerea in pear orchards in the Western Cape of South Africa

Botrytis cinerea isolates from pear blossoms (Pyrus communis) in South Africa were collected from four orchards in two production areas in the Western Cape. The cryptic species status based on vegetative‐incompatibility alleles of the Bc‐hch gene indicated that all the isolates belonged to B. cinerea. A microsatellite analysis of B. cinerea populations was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Some genotype flow was evident between orchards as indicated by the spread of microsatellite multilocus genotypes, in agreement with the moderate, but significant population differentiation among orchards (mean φ_PT = 0.118, P = 0.001). Index of association analyses (I_A and r?_d) suggest that the populations reproduce mostly asexually, even though mating type distribution did not differ significantly from a 1:1 ratio, suggesting frequency‐dependent selection. Isolates resistant to benomyl were evident in one orchard only. This orchard was also significantly differentiated from all other populations, suggesting infrequent localized selection for benomyl resistance. Overall, the findings of this study highlight the dangers of a mixed reproduction system, and stress the importance of regularly monitoring fungicide resistance levels towards developing more efficient management practices.     

High genotypic and virulence diversity in Ilyonectria liriodendri isolates associated with black foot disease in New Zealand vineyards

A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species‐specific PCR and DNA sequencing from a collection of 174 Ilyonectria‐like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP‐PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour‐joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra‐ and inter‐vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non‐self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1‐year‐old potted vines showed variability in virulence; however, no correlations were detected.     

Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe

The genetic variation and evolutionary mechanisms shaping Cucumber vein yellowing virus (CVYV) populations were investigated by analysis of nucleotide sequences coding for P1b, P1b/P3 and coat proteins (CP) from isolates collected in different countries. The complete genome sequence of isolate ISM from Israel was also determined and compared to those of isolates Jor from Jordan and ALM32 from Spain. This isolate had overall nucleotide identities of 94·23 and 94·96% with ALM32 and Jor, respectively. Nucleotide variation among isolates was not homogeneously distributed, with the 5′ half of the genome being more variable than the 3′ half. A Bayesian phylogenetic tree of the CP showed that CVYV isolates clustered into two main clades: isolates from the Middle East region (Lebanon, Israel and Jordan) clustered in both clades whereas the isolate from Tunisia clustered in clade I and the European isolates clustered as a homogeneous phylogroup in Clade II. A similar topology was observed for P1b but with incongruences with respect to the CP, suggesting genetic exchange among virus isolates, which were confirmed with recombination algorithms. The low genetic diversity within the European phylogroup with respect to the other isolates, neutralist tests and genetic differentiation analyses suggest that the Middle East region is the cradle of CVYV and that a unique virus introduction event occurred in Europe, where the virus spread rapidly. Taken together, these findings indicate a risk of emergence of virulent CVYV isolates in Europe either through migration from the Middle East or by genetic changes of the European isolates.     

Use of nitrate non‐utilizing (nit) mutants to determine phenological stages at which Botrytis cinerea infects wine grapes causing botrytis bunch rot

Botrytis bunch rot (BBR), caused by Botrytis cinerea, degrades wine grapes during ripening, even though infection can occur as early as flowering. Effective BBR management requires knowledge of whether some stages of fruit development are more important than others in relation to infection and BBR severity at harvest. Bunches of Vitis vinifera ‘Sauvignon blanc’ and/or ‘Pinot noir’ were inoculated in two vineyard trials and one glasshouse trial with nitrate non‐utilizing (nit) mutant strains at three phenological stages: early flowering, pre‐bunch closure (PBC) and veraison. Isolates recovered from symptomless berries at veraison and from bunches with symptoms at harvest were screened to measure the incidence of the nit strains used in the inoculations. It was found that latent infections, which resulted in BBR at harvest, could become established at all three phenological stages and no single stage was associated with greater latent incidence or harvest severity than any other stage. It was concluded that a proportion of BBR at harvest resulted from the expression of latent infections that had accumulated throughout the season. However, the time between infection and BBR symptom expression in near‐ripe grape berries was sufficiently short for polycyclic secondary infection to also contribute to epidemic development.     

High genetic and virulence diversity detected in Neofusicoccum luteum and N. australe populations in New Zealand vineyards

Genotypic and virulence diversity of Neofusicoccum luteum and N. australe isolates recovered from grapevines displaying symptoms of dieback and decline in New Zealand were investigated. The universally primed PCR (UP‐PCR) method was used to investigate the genetic diversity of 40 isolates of N. luteum and 33 isolates of N. australe. Five UP‐PCR primers produced a total of 51 loci from N. luteum and 57 from N. australe with a greater number of polymorphic loci produced in N. australe (86%) compared with N. luteum (69%). Analysis of UP‐PCR data showed both species found in New Zealand vineyards were genetically diverse at both the inter‐ and intra‐vineyard levels with only a single pair of clonal isolates in N. luteum. Cluster analysis of UP‐PCR data produced four genetic groups in N. luteum and 10 in N. australe (P < 0.05). For both species, there was no relationship between the genetic groups and the origin of isolates. The mean genetic diversity (H) of N. luteum was less than for N. australe, being 0.1791 and 0.2417, respectively. Pathogenicity assays of both species using isolates from either the same or different genetic groups inoculated onto either green shoots or grapevine trunks, showed virulence diversity within the population; however, no correlation was identified between genetic groups and virulence.     

北京地区草莓灰霉病菌的转座子及其分布频率

为了解北京地区草莓灰霉病菌的转座子类型及其分布,本研究用转座子Boty和Flipper的特异性引物对北京地区2012-2013年从12个草莓园采集和分离的60株草莓灰霉病菌进行PCR扩增。结果表明,北京地区草莓灰霉病菌群体中共存在3种转座子类型:transposa型、Boty型和Flipper型。其中,以transposa型菌株最多,占供试菌株的63.3%,Boty型菌株占供试菌株的28.3%,Flipper型菌株最少,仅占8.4%,未检测到vacuma型菌株。选取属于不同转座子类型的18株菌株测定其对草莓叶片的致病力,结果显示Boty型菌株所致病斑的平均直径显著大于Flipper型。草莓灰霉病菌转座子类型与致病力的关系有待进一步研究。转座子类型的检测为进一步研究灰葡萄孢的遗传多样性及遗传变异提供了基础。     

Role of strawberry volatile organic compounds in the development of Botrytis cinerea infection

Botrytis cinerea, the main pathogen of strawberry, has the ability to remain quiescent in unripe tissue and develop disease symptoms in ripe fruit. As strawberry ripening is characterized by an increase of aroma compounds, the role of volatile emission in the development of infection was investigated. Thirty‐five strawberry volatile organic compounds (VOCs) were tested on B. cinerea in vitro and volatile emission was analysed in strawberry harvested at four ripening stages by headspace solid‐phase microextraction/gas chromatography–mass spectrometry and proton transfer reaction–time of flight–mass spectrometry. The coupling of such data sets made it possible to conclude that key strawberry aroma compounds stimulate B. cinerea conidial germination and some typical wound‐volatiles stimulate pathogen conidial germination or mycelial growth. This study is the first report of fungal stimulation by some VOCs naturally occurring in strawberry: the esters ethyl butanoate, cis‐3‐hexenyl acetate, trans‐2‐hexenyl acetate, methyl butanoate and hexyl butanoate, the furanones furaneol and mesifurane, and the alcohol trans‐2‐hexenol. The results of this work provide advances in understanding the functional role of fruit VOCs and suggest, for the first time, that fruit VOCs may influence the development of B. cinerea from the latent phase and that they could favour the invasive growth of B. cinerea after wounding. In particular, ethyl butanoate and furaneol could signal strawberry ripening, and the green leaf volatiles trans‐2‐hexenol, trans‐2‐hexenyl acetate and cis‐3‐hexenyl acetate could signal the presence of damaged tissues that are easier sites for penetration by B. cinerea.     

Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum

The effect of soil solarization and Trichoderma harzianum on induced resistance to grey mould (Botrytis cinerea) and powdery mildew (Podosphaera xanthii) was studied. Plants were grown in soils pretreated by solarization, T. harzianum T39 amendment or both, and then their leaves were inoculated with the pathogens. There was a significant reduction in grey mould in cucumber, strawberry, bean and tomato, and of powdery mildew in cucumber, with a stronger reduction when treatments were combined. Bacillus, pseudomonad and actinobacterial communities in the strawberry rhizosphere were affected by the treatments, as revealed by denaturing gradient gel electrophoresis fingerprinting. In tomato, treatments affected the expression of salicylic acid (SA)‐, ethylene (ET)‐ and jasmonic acid (JA)‐responsive genes. With both soil treatments, genes related to SA and ET – PR1a, GluB, CHI9 and Erf1 – were downregulated whereas the JA marker PI2 was upregulated. Following soil treatments and B. cinerea infection, SA‐, ET‐, and JA‐related genes were globally upregulated, except for the LOX genes which were downregulated. Upregulation of the PR genes PR1a, GluB and CHI9 in plants grown in solarized soil revealed a priming effect of this treatment on these genes' expression. The present study demonstrates the capacity of solarization and T. harzianum to systemically induce resistance to foliar diseases in various plants. This may be due to either a direct effect on the plant or an indirect one, via stimulation of beneficial microorganisms in the rhizosphere.     

Genetic diversity and pathogenicity of Monilinia polystroma – the new pathogen of cherries

Brown rot caused by fungi belonging to the genus Monilinia is one of the major limiting factors of sour and sweet cherry production. Up to now, three species, M. fructigena, M. laxa and M. fructicola, have been identified as causal agents of brown rot on cherries worldwide. From 2010 to 2013, during the monitoring of cherry orchards in different areas of Poland, a fourth species, M. polystroma, was isolated from brown rot symptoms on sour and sweet cherry fruits. To the best of the authors’ knowledge, this is the first time M. polystroma has been reported as the causal agent of brown rot on cherries. The genetic diversity of M. polystroma isolates from cherries and other hosts was analysed using PCR MP, ISSR and RAPD techniques and showed its clear distinctness from other Monilinia spp. tested. The cluster analysis of fingerprinting data revealed a high similarity of M. polystroma isolates from Poland and their close relationship with the reference strain from Japan, indicating that this species is a recently introduced pathogen. The highest genetic distance between the examined isolates and the highest number of different genotypes was observed in an ISSR assay. Detailed genetic diversity characteristics revealed that M. polystroma isolates from cherries did not create a distinct group but were intermingled with M. polystroma isolates from other hosts. The results of the pathogenicity test conducted on different fruit species indicated a lack of host specificity for M. polystroma isolates.     

Genetic diversity and structure of Hemileia vastatrix populations on Coffea spp.

Coffee leaf rust is the most limiting disease for coffee cultivation in Brazil. Despite its importance, relatively little is known about the genetic diversity of Hemileia vastatrix, the rust causal agent. In this work, the DNA from 112 monopustule isolates from different geographic locations and coffee genotypes were analysed by amplified fragment length polymorphisms (AFLP). The objectives were to assess the influence of the host and geographic origin on the diversity and population differentiation in H. vastatrix. The fungal population showed a low level of genotypic diversity. Gene diversity (h) was 0·027 and the hypothesis of random mating in the total population was rejected, but evidence for recombination was found for two subpopulations (São Paulo and Paraná). The analysis of molecular variance revealed that 90% of the genetic distribution of the pathogen occurs among isolates within the subpopulation (states or host of origin). There was no correlation between geographic and genetic distance (r = ?0·024, P = 0·74), which together with the high number of migrants and the low degree of differentiation in populations of H. vastatrix, is consistent with the fact that the inoculum is probably easily dispersed by wind over long distances, allowing dispersal of the pathogen among coffee growing areas in Brazil. Therefore, it is difficult to predict the durability of resistant sources to coffee rust. The recommendation for the breeding programmes is thus to incorporate multigenic resistance as a control strategy.     

Genetic diversity of giant reed ( A rundo donax) in Australia

The perennial grass, Arundo donax, has shown potential as a promising biomass crop. However, it has become invasive in a number of areas and declared a noxious weed in some jurisdictions, making proposals to grow A. donax for commercial use in Australia controversial. Evidence of asexual reproduction and the presence of a single genetic clone in Australia was investigated, as such characteristics would indicate a limited risk of escape and invasion. Using amplified fragment length polymorphism markers, the genetic diversity of 218 A. donax samples from across Australia was examined. The samples were found to separate into two distinct genetic groups, or clades. There was only a small amount of genetic diversity within a clade (0.9 and 1.5%). However, there was a larger difference between the clades of 19.8%, suggesting the presence of two distinct A. donax genotypes in Australia. The low level of genetic variation in Australian A. donax that was found in this study indicates that spread is essentially by vegetative means and suggests that if grown in areas where it is separated from natural water dispersal events, A. donax poses a low risk of becoming invasive.     

Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination

The biological and molecular characterization of six isolates of a new Cowpea mild mottle virus strain (CPMMV; Carlavirus, Betaflexiviridae) are reported. Soybean plants with mosaic and stem necrosis were collected in Bahia, Goiás, Mato Grosso and Minas Gerais states, Brazil. Complete genomes of the CPMMV isolates are 8180–8198 nucleotides (nt) long, excluding the 3′‐polyadenylated tail, and have 67–68% nt sequence identity with a Ghana isolate of CPMMV, the only CPMMV isolate for which the genome has previously been sequenced. The replicase has only 60–61% nt sequence identity with the Ghana CPMMV isolate, and the coat protein (CP) is highly conserved (79% nt sequence identity and 95–96% amino acid sequence identity). The high CP identity and the phylogenetic analyses supported the classification of the Brazilian isolates as CPMMV. Biological and molecular differences with the Ghana CPMMV isolate were found and indicated that the six isolates represent a distinct CPMMV strain denominated as CPMMV‐BR. Furthermore, it is shown that recombination occurred mainly in the polymerase gene, and may occur less frequently in other regions of the CPMMV genome.     

Genetic variation and structure in native and invasive Solidago canadensis populations

Solidago canadensis is native to North America, but has become a noxious invasive plant in China. We know only a little about its invasion history and the effects of introductions on its genetic composition. Here, we investigated genetic variation and structure between 15 North American and 13 Chinese populations of S. canadensis using AFLP makers. Four AFLP loci suggested relatively high genetic diversity of this weed and similar genetic variation between the invasive range and the native range. Most genetic variation was within populations across two ranges, but the Chinese range had a higher degree of among‐population variation than the North American range. Multiple tests, including Bayesian assignment, UPGMA analysis, PCoA and analysis of ‘isolation by distance’, showed that the Chinese populations originated from at least two distinct native sources and that secondary introduction or dispersal should be common in China. Also, North American populations were possibly a single genetic group. Overall, S. canadensis in China was probably founded from multiple introductions and then spread through long‐distance dispersal associated with human activities. Genetic variability in the species in the invaded range appears to have favoured establishment and spread and may well provide a challenge to successful control.     

Pathogenic variation of Mycosphaerella species infecting banana and plantain in Nigeria

Mycosphaerella species that cause the ‘Sigatoka disease complex’ account for significant yield losses in banana and plantain worldwide. Disease surveys were conducted in the humid forest (HF) and derived savanna (DS) agroecological zones from 2004 to 2006 to determine the distribution of the disease and variation among Mycosphaerella species in Nigeria. Disease prevalence and severity were higher in the HF than in the DS zone, but significant (P < 0·001) differences between agroecological zones were only observed for disease severity. A total of 85 isolates of M. fijiensis and 11 isolates of M. eumusae were collected during the survey and used to characterize the pathogenic structure of Mycosphaerella spp. using a putative host differential cultivar set consisting of Calcutta‐4 (resistant), Valery (intermediate) and Agbagba (highly susceptible). Area under disease progress curve (AUDPC) was higher on all cultivars when inoculated with M. eumusae than with M. fijiensis, but significant (P < 0·05) differences between the two species were only observed on Valery. Based on the rank‐sum method, 8·3% of the isolates were classified as highly aggressive and 46·9% were classified as aggressive. About 11·5% of all the isolates were classified as least aggressive, and all of these were M. fijiensis. The majority of M. eumusae isolates (seven out of 11; 64%) were classified as aggressive. A total of nine pathotype clusters were identified using cluster analysis of AUDPC. At least one M. fijiensis isolate was present in all the nine pathotype clusters, while isolates of M. eumusae were present in six of the nine clusters. Isolates in pathotype clusters III and V were the most aggressive, while those in cluster VIII were the least aggressive. Shannon’s index (H) revealed a more diverse Mycosphaerella collection in the DS zone (H = 1·81) than in the HF (H = 1·50) zone, with M. fijiensis being more diverse than M. eumusae. These results describe the current pathotype structure of Mycosphaerella in Nigeria and provide a useful resource that will facilitate screening of newly developed Musa genotypes for resistance against two important leaf spot diseases of banana and plantain.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

The intriguing and convoluted life of a heteroecious rust fungus in New Zealand

Molecular phylogenetic analyses of New Zealand rust fungi suggested that four taxa, Aecidium otagense on Clematis spp., Puccinia tiritea on Muehlenbeckia spp., Puccinia rhei‐undulati sensu auct. NZ, non (Dietel) Hirats. f. on Rheum ×rhabarbarum, and an unidentified Puccinia species on Rumex sagittatus, are a single species. Morphological studies and multilocus molecular data, together with inoculation studies, confirmed this finding. This species is only the third heteroecious rust fungus known to be native to New Zealand.     

The morphology,behaviour and molecular phylogeny of Phytophthora taxon Salixsoil and its redesignation as Phytophthora lacustris sp. nov.

Since its first isolation from Salix roots in 1972, isolates of a sexually sterile Phytophthora species have been obtained frequently from wet or riparian habitats worldwide and have also been isolated from roots of Alnus and Prunus spp. Although originally assigned to Phytophthora gonapodyides on morphological grounds, it was recognized that these isolates, informally named P. taxon Salixsoil, might represent a separate lineage within ITS Clade 6. Based on phylogenetic analyses and comparisons of morphology, growth‐temperature relationships and pathogenicity, this taxon is formally described here as Phytophthora lacustris sp. nov. Isolates of P. lacustris form a clearly resolved cluster in both ITS and mitochondrial cox1 phylogenies, basal to most other Clade 6 taxa. Phytophthora lacustris shares several unusual behavioural properties with other aquatic Clade 6 species, such as sexual sterility and tolerance of high temperatures, that have been suggested as adaptations to riparian conditions. It appears to be widespread in Europe and has also been detected in Australia, New Zealand and the USA. It was shown to be weakly or moderately aggressive on inoculation to Alnus, Prunus and Salix. The extent of P. lacustris’ activity as a saprotroph in plant debris in water and as an opportunistic pathogen in riparian habitats needs further investigation. Its pathogenic potential to cultivated fruit trees also deserves attention because P. lacustris has apparently been introduced into the nursery trade.