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.     

Phakopsora myrtacearum sp. nov., a newly described rust (Pucciniales) on eucalypts in eastern and southern Africa

Outbreaks of a rust disease in eucalypt forestry plantations and nurseries in Kenya, Mozambique and South Africa occurred between 2009 and 2014. The pathogen was identified using morphology and molecular phylogenetic analyses as an undescribed species in the Phakopsoraceae. A systematic study, based on nuclear ribosomal DNA, showed that it is a species of Phakopsora, herein named Phakopsora myrtacearum sp. nov. This new species of rust is the second validly described species on Eucalyptus, along with Puccinia psidii. Phakopsora myrtacearum is distinguished from P. psidii by leaf symptoms, morphology of the urediniospores and distinct phylogenetic placement. Phakopsora myrtacearum has been found on three species of Eucalyptus in Kenya, Mozambique and South Africa, and it may have future negative implications for commercial forestry in these areas.     

Phytophthora oleae sp. nov. causing fruit rot of olive in southern Italy

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.     

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1‐α (EF1‐α) and β‐tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania.     

Phytophthora caryae sp. nov., a new species recovered from streams and rivers in the eastern United States

Members of the Phytophthora citricola complex (Phytophthora clade 2c), such as P. plurivora, are destructive pathogens of trees and shrubs in nursery, landscape and forest settings worldwide. During surveys of Phytophthora species from streams and rivers in Massachusetts and North Carolina, a novel species in the P. citricola complex was recovered. Based on sequences from three nuclear (ITS, β‐tub and tef1) and two mitochondrial (cox1 and nadh1) loci, morphological characters, temperature–growth relationships and host plant inoculations, this novel species is described as Phytophthora caryae sp. nov. Phytophthora caryae resembles several other species in the P. citricola complex, demonstrating homothallism and producing paragynous antheridia and semipapillate and noncaducous sporangia. However, P. caryae exhibits smaller sexual structures, higher rates of oogonia with a tapered base and sporangia with an offset attachment of the sporangiophores. Phylogenetic analyses using maximum likelihood and Bayesian inference placed isolates of P. caryae into a unique clade with significant statistical support. Based on the mitochondrial dataset, P. caryae is most closely related to P. pini and P. citricola III, which are believed to be native in eastern North America. Inoculations of P. caryae on 1‐year‐old twigs of 12 tree species representing nine genera resulted in under‐bark lesions on species of Carya and Juglans. Sapling inoculations under greenhouse conditions suggest that P. caryae may be pathogenic to shagbark hickory (Carya ovata) but not to black walnut (Juglans nigra).     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Characterization and taxonomic reassessment of the box blight pathogen Calonectria pseudonaviculata,introducing Calonectria henricotiae sp. nov.

Calonectria pseudonaviculata, the causal agent of the disease of Buxus spp. known as ‘box blight’, was first detected in the mid‐1990s in the UK and New Zealand. Since then, the geographic range of box blight has rapidly expanded to at least 21 countries throughout temperate regions of the world, causing significant losses in nurseries, gardens and wild boxwood populations. This study determined the genetic diversity in a collection of 234 Calonectria isolates from diseased Buxus plants, originating from 15 countries and four continents. Two genetic clades, G1 and G2, were identified within this sample using multilocus phylogenetic analysis. The application of genealogical concordance phylogenetic species recognition criteria using four independent nuclear loci determined that the Calonectria isolates in these two clades are separate phylogenetic species. The isolates in the G1 clade were upheld as C. pseudonaviculata sensu stricto. Based on phylogenetic distinctiveness and the lack of mating, a new species is proposed, Calonectria henricotiae sp. nov., for the Calonectria isolates in the G2 clade. A PCR‐RFLP assay and real‐time PCR assays were developed to easily and reproducibly differentiate these species. To assess the practical implications of the identification of the two species, their physiology, fungicide susceptibility and pathogenicity were compared. No differences in pathogenicity were observed. However, C. henricotiae isolates exhibited greater thermotolerance and reduced sensitivity to specific triazole as well as strobilurin fungicides. The identification of a second phylogenetic species causing box blight may have a substantial impact on the epidemiology and control of this destructive disease.     

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.     

A new anthracnose disease of pyrethrum caused by Colletotrichum tanaceti sp. nov

A new pathogen of pyrethrum (Tanacetum cinerariifolium) causing anthracnose was described as Colletotrichum tanaceti based on morphological characteristics and a four‐gene phylogeny consisting of rDNA‐ITS, β‐tubulin (TUB2), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and actin (ACT) gene sequences. The fungus produced perithecia in culture, requiring an opposite mating type isolate in a heterothallic manner. The initial infection strategy on pyrethrum leaves involved the formation of appressoria followed by production of multilobed infection vesicles in the epidermal cells. Infection and colonization then proceeded through thinner secondary hyphae, which resulted in the initial production of water‐soaked lesions followed by black necrotic lesions. The infection process was suggestive of a hemibiotrophic infection strategy. Moreover, phylogenetic analysis clearly showed that C. destructivum, C. higginsianum and C. panacicola were separate species that also had similar intracellular hemibiotrophic infection strategies as C. tanaceti, which all clustered in the C. destructivum complex. Colletotrichum spp. were detected at 1% incidence in seed of 1 of 19 seed lines, indicating the potential for seed as a source of inoculum into crops. Colletotrichum tanaceti was detected in leaf lesions from 11 of 24 pyrethrum fields surveyed between April and July 2012, at a frequency of 1·3–25·0% of lesions. Anthracnose probably contributes to the complex of foliar diseases reducing green leaf area in pyrethrum fields in Australia.     

Development,characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. In this study, an Australian Pga isolate was sequenced, the abundance of simple sequence repeats (SSRs) was determined and PCR‐based polymorphic markers suitable for genetic diversity analysis were developed. The amplification of 194 primer pairs was initially assessed using a set of 12 isolates of different cereal rust species and their formae speciales. A high frequency of cross‐species amplification was observed for most markers; however, 36 SSRs were diagnostic for P. graminis only. A subset of 19 genome‐derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates, previously shown to undergo sexual recombination, were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two subpopulations. The genome‐derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species.     

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

The rust fungi Melampsora larici‐populina (Mlp) and Melampsora allii‐populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co‐infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real‐time PCR assay targeting each species was developed, based on single‐copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time‐course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co‐inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

Frequency of brown rot fungi on blossoms and fruit in stone fruit orchards in Greece

Brown rot is a devastating disease of stone fruits caused by Monilinia spp. This study was conducted to investigate the disease aetiology on blossoms and fruit in peach, apricot, sweet cherry and plum orchards, in Greece. In total, 1433 isolates obtained from orchards located in the main stone fruit production regions of Greece were identified to species based on the presence/size of a cyt b intron. Monilinia laxa and M. fructicola were detected at frequencies of 59 and 41%, respectively, while M. fructigena was absent. Monilinia fructicola was more common on fruit whereas M. laxa occurred in similar frequency on blossoms and fruit. Monilinia laxa was replaced by M. fructicola in fruit infections of peach in both regions investigated and in fruit infections of plum in the Imathia region. Assessments of aggressiveness of 30 isolates of both species on the petals and fruits of the hosts showed that M. fructicola isolates were more aggressive. This suggests that the predominance of M. laxa on the blossoms cannot be explained by higher aggressiveness. Measurements of the effect of temperature on mycelial growth showed that M. laxa isolates had a higher growth rate than M. fructicola at the lowest temperature tested of 5°C, whereas M. fructicola isolates showed higher growth rates at higher temperatures. The observed high frequency of M. fructicola in Greece represents a major threat for stone fruit production. Furthermore, the information obtained about delineation of species and plant organ preference could be useful for the implementation of disease management strategies.     

Overwintering and epidemiology of Puccinia dracunculina,the causal agent of rust in open tarragon fields

Rust, caused by Puccinia dracunculina, is the main foliar disease of open‐field tarragon (Artemisia dracunculus) crops in Israel. As not much is known about the biology or epidemiology of this pathogen, the long‐term objective of the current study was to accumulate the knowledge needed to develop an effective, environmentally friendly means of adequately managing the disease. Puccinia dracunculina is an autoecious brachy‐form pathogen, but it is not known whether the life cycle is completed under field conditions in Israel. Field observations and greenhouse studies revealed that although the telial stage is produced, the pathogen overwinters in the uredinial stage. In vitro experiments were used to quantify the temperature and wetness requirements for urediniospore germination and to calculate the daily duration of conducive weather (DDCW); DDCW was defined as the number of hours during which temperature ranged between 15 and 25°C and RH was >90%. Cumulative DDCW values (CDDCW) were a good predictor of disease under natural conditions in two growing seasons. Disease onset occurred when CDDCW values reached a level of 10 and the relationship between log CDDCW values and season‐long severity values (in logit) was highly significant, explaining 90·6% of the variation.     

A Tobacco etch virus NW isolate that overcomes pvr1 and pvr12 resistance in Capsicum sp.

Two important sources of Capsicum annuum (bell pepper) resistance were evaluated for their response to inoculation with two isolates of Tobacco etch virus strain NW (TEV‐NW, genus Potyvirus). The resistant cultivars were CA4 and Dempsey, which contain the pvr1 and pvr1² resistance genes, respectively. TEV‐NW was maintained by mechanical passage in the susceptible pepper cultivar Early Calwonder and Nicotiana tabacum cv. Kentucky 14. In initial experiments, the TEV‐NW isolate maintained in Early Calwonder infected two of seven CA4 plants; however, none of the CA4 plants inoculated with the TEV‐NW isolate maintained in Kentucky 14 were infected. The infected CA4 plants had low virus titres in non‐inoculated leaves and did not develop visible symptoms. When the infected CA4 plants were used as inoculum of additional CA4 plants, all newly inoculated plants became infected, developed systemic symptoms and accumulated virus in non‐inoculated leaves more quickly than the originally infected CA4 plants. This new NW isolate, referred to as NW‐CA4, was shown to overcome the resistances expressed by both CA4 (pvr1) and Dempsey (pvr1²). The potyviral VPg is believed to be the determinant for pvr1 and pvr1² resistance genes, both of which are eIF4E‐encoding genes. The VPg amino acid sequence for NW‐CA4 was determined and compared with that of NW isolates and different TEV strains. No amino acid variation was identified that explained the infectivity of NW‐CA4 in CA4 and Dempsey plants.     

Local dispersal of Puccinia striiformis f. sp. tritici from isolated source lesions

Understanding how disease foci arise from single source lesions has not been well studied. Here, single wheat leaves were inoculated with Puccinia striiformis f. sp. tritici urediniospores, and all wheat leaves within two intersecting 0.3 × 3.0 m transects were sampled in eight replicates over 3 years. The lesions observed on each of the top three leaves on plants within 1.5 m from the source lesion were three‐dimensionally mapped. The total number of lesions within a 1.5 m radius was estimated by dividing the number of lesions observed within each 0.025 m‐wide annulus by the fraction of the annulus sampled. The estimated total number of lesions produced within 1.5 m of a single source lesion ranged from 27 to 776, with a mean of 288 lesions. Eighty percent of the lesions were recorded within 0.69 m of the source infection. The proportion of total lesions observed at a given distance from the source was fitted well by the Lomax and Weibull distributions, reflecting the large proportion of lesions arising close to the source, and when fitted by an inverse power distribution had a slope (b) of 2.5. There were more lesions produced on leaves higher in the canopy than on lower leaves, with more lesions being detected above than below the point of inoculation. Simultaneous measurement of lesion gradients and spore dispersal in the final year of the study suggests that this pattern is due to greater susceptibility of upper leaves, rather than increased dispersal to upper leaves.     

Mapping of Aegilops umbellulata‐derived leaf rust and stripe rust resistance loci in wheat

Aegilops umbellulata, a non‐progenitor diploid species, is an excellent source of resistance to various wheat diseases. Leaf rust and stripe rust resistance genes from A. umbellulata were transferred to the susceptible wheat cultivar WL711 through induced homoeologous pairing. A doubly resistant introgression line IL 393‐4 was crossed with wheat cultivar PBW343 to develop a mapping population. Tests on BC₂F₇ RILs indicated monogenic inheritance of seedling leaf rust and stripe rust resistance in IL 393‐4 and the respective co‐segregating genes were tentatively named LrUmb and YrUmb. Bulked segregant analysis placed LrUmb and YrUmb in chromosome 5DS, 7.6 cM distal to gwm190. Aegilops geniculata‐derived and completely linked leaf rust and stripe rust resistance genes Lr57 and Yr40 were previously located in chromosome 5DS. STS marker Lr57/Yr40MAS‐CAPS16 (Lr57/Yr40‐CAPS16), linked with Lr57/Yr40 (T756) also co‐segregated with LrUmb/YrUmb. Seedling infection types differentiated LrUmb from Lr57. Absence of leaf rust‐susceptible segregants among F₃ families of the intercross (IL 393‐4/T756) indicated repulsion linkage between LrUmb and Lr57. YrUmb expressed a consistently low seedling response under greenhouse conditions, whereas Yr40 expressed a higher seedling response. Based on the origin of LrUmb/YrUmb from the U genome and Lr57/Yr40 from the M genome, as well as phenotypic differences, LrUmb and YrUmb were formally named Lr76 and Yr70, respectively. These genes have been transferred to Indian wheat cultivars PBW343 and PBW550, and advanced breeding lines are being tested in state and national trials.     

Stem rot of jewel orchids caused by a new forma specialis,Fusarium oxysporum f. sp. anoectochili in Taiwan

Stem rot of Anoectochilus formosanus (Af) caused by Fusarium oxysporum (Fo) is a major limiting factor to jewel orchid production in Taiwan. Fo causes discoloration in vascular tissues. However, some newly collected Fo isolates from Af stem rot do not cause vascular discoloration, suggesting changes may have occurred in the pathogen. Among recent Fo isolates from Af there are two colony types, the cottony alba (CA) and the sporodochial (S). In order to confirm that both colony types cause Af stem rot, 200 isolates were obtained from diseased stems in Nantou County and characterized by colony type and whether or not the infected plants had vascular discoloration. Isolates of both the CA and S types caused stem rot of Af; some isolates in each colony type caused vascular discoloration whilst others did not. Pathogenicity tests with 22 isolates resulted in stem rot disease severity ratings on Af of 3·1–4·0 and 2·1–4·0 with CA and S type colonies, respectively. The same isolates failed to cause disease on Cattleya, Dendrobium or Phalaenopsis plants. Phylogenetic analysis of partial intergenic spacer sequences showed that these isolates were distinguishable from other formae speciales of Fo and could be separated into two groups correlated with the CA or S type colonies with high bootstrap. Based on pathogenic, morphological and molecular characterizations, the Fo that causes stem rot of Af is proposed to be a new forma specialis, F. oxysporum f. sp. anoectochili, with different pathotypes.     

Genetic differentiation of the wheat leaf rust fungus Puccinia triticina in Europe

The objective of this study was to determine whether genetically differentiated groups of Puccinia triticina are present in Europe. In total, 133 isolates of P. triticina collected from western Europe, central Europe and Turkey were tested for virulence on 20 lines of wheat with single leaf rust resistance genes, and for molecular genotypes with 23 simple sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotype within countries, 121 isolates were retained for further analysis. Isolates were grouped based on SSR genotypes using a Bayesian approach and a genetic distance method. Both methods optimally placed the isolates into eight European (EU) groups of P. triticina SSR genotypes. Seven of the groups had virulence characteristics of isolates collected from common hexaploid wheat, and one of the groups had virulence characteristics of isolates from tetraploid durum wheat. There was a significant correlation between the SSR genotypes and virulence phenotypes of the isolates. All EU groups had observed values of heterozygosity greater than expected and significant fixation values, which indicated the clonal reproduction of urediniospores in the overall population. Linkage disequilibria for SSR genotypes were high across the entire population and within countries. The overall values of R_ST and F_ST were lower when isolates were grouped by country, which indicated the migration of isolates within Europe. The European population of P. triticina had higher levels of genetic differentiation compared to other continental populations.