Global movement and population biology of Mycosphaerella nubilosa infecting leaves of cold-tolerant Eucalyptus globulus and E. nitens

Using 10 polymorphic DNA-based microsatellite markers, the genetic diversity of eight Mycosphaerella nubilosa populations from Eucalyptus , comprising 497 isolates from five different countries, was studies using a hierarchical sampling regime. Mycosphaerella nubilosa from eastern Australia (New South Wales) had higher gene (0·506) and genotypic (76%) diversity than other populations, supporting the view that this represents the origin of the pathogen. It was also evident that M. nubilosa populations from Europe and Tanzania were clonal, with the same multilocus haplotypes occurring in South Africa, but being absent in Australia. This suggests that M. nubilosa may have been introduced into Europe via Africa, with a pathway of gene flow from Australia to South Africa, further into Africa and finally to Europe.     

Genetic diversity and aggressiveness of Erwinia psidii on Eucalyptus spp. in Brazil

The genetic variability and aggressiveness of Brazilian Erwinia psidii isolates from Eucalyptus spp. was studied and compared with reference isolates from guava (Psidium guajava). Repetitive element sequence (rep)-based PCR markers of 101 isolates from Eucalyptus spp. and five from guava showed that the populations of E. psidii displayed a relatively low genetic variability. No correlation of genetic clustering based on rep-PCR analysis with geographic origin or host of origin was observed, indicating that genome rearrangements associated with adaptation to a particular host were not detected by these molecular markers. A higher genotypic richness was detected in the Mato Grosso do Sul population, probably reflecting a pathogen dissemination associated with the recent expansion in eucalypt plantations. Wilcoxon and ANOVA tests of disease severity data indicated differences in aggressiveness among isolates and an isolate × clone interaction. The area under the disease progress curve (AUDPC) and disease severity for some isolates were significantly different between two susceptible clones tested. Notably, isolate LPF681 from guava was not able to cause disease on a susceptible Eucalyptus urophylla clone, suggesting that some co-evolution between pathogen and host has taken place. The variability in aggressiveness and virulence among isolates of E. psidii observed in this study will be important for the establishment of appropriate screening approaches to select for disease resistance.     

Movement of genotypes of Ceratocystis fimbriata within and among Eucalyptus plantations in Brazil

Ceratocystis wilt on eucalyptus, caused by Ceratocystis fimbriata, was first recognized in 1997 in the state of Bahia, Brazil, but is now known in five other states and in four other countries. C. fimbriata is a native, soilborne pathogen in some parts of Brazil but we hypothesized that genotypes of the pathogen have been moved among plantations in rooted cuttings collected from diseased trees and within plantations on cutting tools. We used six microsatellite markers to identify 78 genotypes of C. fimbriata among 177 isolates from individual trees in 20 eucalyptus plantations. The highest gene and genotypic diversity values were found in plantations on formerly wild Cerrado forest in Minas Gerais, suggesting that the fungus was in the soil prior to planting eucalyptus. In contrast, one or only a few genotypes were found in plantations on previous pastureland (with no woody hosts) in Bahia and S?o Paulo, and most of these genotypes were found in a Bahian nursery or in one of two Bahian plantations that were sources for rooted cuttings. Sources of cuttings tended to be dominated by one or a few genotypes that may have been spread within the plantation on cutting tools.     

Development of Nested Polymerase Chain Reaction Detection of Mycosphaerella spp. and Its Application to the Study of Leaf Disease in Eucalyptus Plantations

ABSTRACT Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm(2) or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.     

Pathogenic Variation in Colletotrichum gloeosporioides Infecting Stylosanthes spp. in a Center of Diversity in Brazil

ABSTRACT Pathogenic variation in Colletotrichum gloeosporioides infecting species of the tropical pasture legume Stylosanthes at its center of diversity was determined from 296 isolates collected from wild host population and selected germ plasm of S. capitata, S. guianensis, S. scabra, and S. macrocephala in Brazil. A putative host differential set comprising 11 accessions was selected from a bioassay of 18 isolates on 19 host accessions using principal component analysis. A similar analysis of anthracnose severity data for a subset of 195 isolates on the 11 differentials indicated that an adequate summary of pathogenic variation could be obtained using only five of these differentials. Of the five differentials, S. seabrana 'Primar' was resistant and S. scabra 'Fitzroy' was susceptible to most isolates. A cluster analysis was used to determine eight natural race clusters using the 195 isolates. Linear discriminant functions were developed for eight race clusters using the 195 isolates as the training data set, and these were applied to classify a test data set of the remaining 101 isolates. All except 11 isolates of the test data set were classified into one of the eight race clusters. Over 10% of the 296 isolates were weakly pathogenic to all five differentials and another 40% were virulent on just one differential. The unclassified isolates represent six new races with unique virulence combinations, of which one isolate is virulent on all five differentials. The majority of isolates came from six field sites, and Shannon's index of diversity indicated considerable variation between sites. Pathogenic diversity was extensive at three sites where selected germ plasm were under evaluation, and complex race clusters and unclassified isolates representing new races were more prevalent at these sites compared with sites containing wild Stylosanthes populations.     

Genetic diversity and aggressiveness of Calonectria pteridis in Eucalyptus spp.

Calonectria leaf blight, caused by Calonectria pteridis, is currently one of the main foliar diseases in eucalypt plantations in Brazil. In warm and high rainfall regions, the disease can be a limiting factor for eucalypt production when planting susceptible genotypes. The most effective method for controlling this disease in the field is the use of resistant genotypes, which requires knowledge of the genetic variability and aggressiveness of the pathogen population for effective deployment of plant resistance. This work evaluated the genetic diversity and aggressiveness of C. pteridis populations obtained from infected eucalypt plants in Monte Dourado (Pará state) and Imperatriz (Maranhão state), Brazil. To study the genetic diversity, 16 ISSR primers were tested, five of which amplified polymorphic, reproducible and informative bands. Thirty-one closely related genotypes were identified from 84 isolates studied, indicating that the population has a low genetic diversity. The aggressiveness of seven isolates, selected according to geographic origin and their clustering in the ISSR-based dendogram, was determined by inoculation of a hybrid Eucalyptus grandis × E. urophylla clone under controlled conditions. Disease severity was assessed by both measuring the percentage of plant defoliation and assigning a score according to a diagrammatic scale of symptoms. A high correlation between the two evaluation methods was observed, which revealed significant differences in aggressiveness among the isolates. The diagrammatic scale is recommended for disease evaluation because results are obtained much faster, before the occurrence of severe defoliation. No correlation between clustering in the ISSR-based phylogenetic analysis and aggressiveness was observed.     

Teratosphaeria pseudoeucalypti,new cryptic species responsible for leaf blight of Eucalyptus in subtropical and tropical Australia

Sub‐tropical and tropical plantations of Eucalyptus grandis hybrids in eastern Australia have been severely affected by anamorphs of Teratosphaeria (formerly Kirramyces) causing a serious leaf blight disease. Initially the causal organism in Queensland, Australia, was identified as Teratosphaeria eucalypti, a known leaf parasite of endemic Eucalyptus spp. However, some inconsistencies in symptoms, damage and host range suggested that the pathogen in Queensland may be a new species. Isolates of T. eucalypti from throughout its known endemic range, including Queensland and New Zealand, where it is an exotic pathogen, were compared using multiple gene phylogenies. Phylogenetic studies revealed that the species responsible for leaf blight in Queensland represents a new taxon, described here as Teratosphaeria pseudoeucalypti. While the DNA sequence of T. pseudoeucalypti was more similar to T. eucalypti, the symptoms and cultural characteristics resembled that of T. destructans. The impact of this disease in central Queensland has increased annually and is the major threat to the eucalypt plantation industry in the region.     

Disease cycle of Austropuccinia psidii on Eucalyptus globulus and Eucalyptus obliqua leaves of different rust response phenotypes

Myrtle rust poses a significant biosecurity threat to Australia with potential for long-term damaging impacts on native flora and plant industries. This study describes the disease cycle of Austropuccinia psidii, the myrtle rust pathogen, in Eucalyptus globulus and Eucalyptus obliqua, two commercially and ecologically important species from different subgenera of Eucalyptus. Ontogeny and morphology of infection structures of A. psidii on plants of both Eucalyptus species with different rust response phenotypes, i.e. completely resistant, hypersensitive and highly susceptible, were investigated. Plants were inoculated with single-uredinium-derived urediniospores and examined by scanning electron microscopy. No differences between rust response phenotypes were observed in germination of urediniospores, formation of appressoria or length of germ tubes. The growth of germ tubes had no affinity towards stomata of either species. Histological observations indicated direct penetration by infection pegs through the leaf cuticle and no penetration beyond the epidermis on rust-resistant E. obliqua. Eucalyptus obliqua plants that were identified as susceptible to A. psidii at 3- and 6-months-old showed no disease when reinoculated with A. psidii at 12-months-old; this indicated possible early acquisition of adult plant resistance to A. psidii in this species. In the susceptible phenotype of E. globulus rust inoculation led to rapid colonization of leaf parenchyma cells with the disease cycle completed within 10 days. These findings provide valuable insights into host–pathogen interactions in the Eucalyptus–A. psidii pathosystem, which might be useful for the development of effective rust control strategies across Eucalyptus subgenera.     

Attractiveness and oviposition preference of Glycaspis brimblecombei Moore in Eucalyptus spp.

The red gum lerp psyllid Glycaspis brimblecombei Moore (Hemiptera: Psyllidae) was first recorded in Brazil in 2003 and since then has caused serious damage in Eucalyptus forests. The damage is mainly due to defoliation, sooty mold and dieback, thus requiring the development of efficient management strategies. The settling and ovipositional preference of G. brimblecombei by species and clones of Eucalyptus were evaluated in free- and no-choice tests. Preliminary tests were done to determine the density of psyllid couples that produced the greatest densities of eggs, leaf position selection for oviposition, and whether oviposition was affected by preimaginal conditioning. Apical leaves were used more frequently for oviposition by the psyllid, and the number of eggs decreased from the apex to the base. The densities of 10 and 15 psyllid couples per seedling produced the highest oviposition. There was no evidence of preimaginal conditioning. In the free-choice test, E. grandis, E. urophylla, VM-1, I-144, C-219 and H-13 were less attractive to G. brimblecombei adults, with H-13, E. grandis and E. urophylla having the least oviposition. In the no-choice test, E. urophylla, GG-100 and E. grandis also demonstrated a lower oviposition, but E. camaldulensis and 3025 were highly susceptible. The low preference for the genotypes E. grandis and E. urophylla suggests the occurrence of a non-preference type resistance against the red gum lerp psyllid. Our results can be utilized for the development of management programs for G. brimblecombei in Eucalyptus forests.     

Low genetic diversity and strong geographic structure in introduced populations of the Eucalyptus foliar pathogen Teratosphaeria destructans

The aggressive Eucalyptus leaf pathogen, Teratosphaeria destructans, causes widespread damage in tropical and subtropical Eucalyptus-growing regions of Indonesia, China, Thailand, East Timor, Vietnam, Lao, and South Africa. Little is known regarding the origin, pathways of dispersal, or reproductive biology of this pathogen. The aim of this study was to investigate the genetic structure of a global collection of T. destructans isolates. This was achieved by developing and using polymorphic microsatellite markers. Low genotypic diversity and a limited number of private alleles were found in all investigated populations, with the highest maximum diversity of 10.7% in isolates from South Sumatra. This supports the hypothesis that T. destructans was introduced to these regions. High levels of clonality were common in all populations, especially in isolates sampled from the recent disease outbreak in South Africa, which were all identical. The global collection of isolates grouped into three distinct clusters, corresponding largely to their sampled regions. Low levels of genotypic diversity, high levels of clonality, and strong geographic structure suggest independent introductions into all the sampled areas from an unknown source. The results imply that strict biosecurity measures are needed to avoid introductions of additional genotypes in these areas.     

Diversity and Ecology of Biocontrol Pseudomonas spp. in Agricultural Systems

ABSTRACT Diverse Pseudomonas spp. may act as biological controls of plant pathogens, but the ecology of those natural populations is not well understood. And, while biocontrol potential has been identified in multiple pseudomonad strains, the linkages between genotype and phenotype have yet to be fully delineated. However, intensive studies of one class of biocontrol strains, i.e., those that can produce 2,4-diacetylphloroglucionl (DAPG), have provided new insights into the diversity, distribution, and interactions of biocontrol pseudomonads. Those studies also laid the foundation for future research and development of pseudomonad-based biocontrol strategies. Over the past several years, numerous studies have also revealed that biocontrol pseudomonads are widely distributed in agricultural soils, and that multiple crop and soil factors can affect their abundance and activities. Recent work has shown that a variety of farm management practices that reduce soilborne disease pressure can also alter the rhizosphere abundance of DAPG producers in complex ways. Such studies provide support for the hypothesis of an ecological feedback mechanism whereby a native biocontrol population increase and subsequently reduce root disease severity following infection. It is well established that complex biological interactions can take place among bio-control pseudomonads, plant pathogens, their hosts, and other members of the microbial community. The net result of such interactions likely dilutes biocontrol efficacy at the field scale. Nonetheless, inoculation can be effective, and several successful applications of biocontrol pseudomonads have been developed. Future applications of microbial ecology research will hopefully improve the consistency and efficacy of bio-control mediated by Pseudomonas spp. Current applications and future opportunities for improving pseudomonad-based biological control are discussed.     

Infection and disease development of Quambalaria spp. on Corymbia and Eucalyptus species

Quambalaria spp. are eucalypt leaf and shoot pathogens of growing global importance, yet virtually nothing is known regarding the manner in which they infect and colonize their hosts. A study of the infection process of Q. pitereka and Q. eucalypti on Corymbia and Eucalyptus species was thus undertaken using light, scanning and transmission electron microscopy after artificial inoculation. Conidial germination was triggered when relative humidity levels exceeded 90% and commenced within 2 h in the presence of free water. Light reduced germination but did not prevent germination from occurring. Conidial germination and hyphal growth occurred on the upper and lower leaf surfaces with penetration occurring via the stomata or wounds on the leaf surface or juvenile stems. There was no evidence of direct penetration of the host. Following penetration through the stomata, Q. pitereka and Q. eucalypti hyphae grew only intercellularly without the formation of haustoria or interaction apparatus, which is characteristic of the order Microstromatales. Instead, the presence of an interaction zone is demonstrated in this paper. Conidiophores arose through stomatal openings producing conidia 7 days after infection.     

蓝桉果实抗稻瘟菌成分的分离及其活性

 通过抗菌活性测定, 蓝桉(Eucalyptus globulus Labill.)果实乙醇提取物中抗稻瘟菌活性成分主要存在于石油醚萃取部分和乙酸乙酯萃取部分。从石油醚部分和乙酸乙酯部分分离到的15个化合物中, β-桉叶油醇和β-谷甾醇对稻瘟菌孢子萌发具有较强的抑制活性, 它们对稻瘟菌孢子萌发的半抑制浓度(IC₅₀)分别为62.9和141.4μg/mL。其它化合物对稻瘟菌孢子萌发只有轻微的抑制活性。     

Drought × disease interaction in Eucalyptus globulus under Neofusicoccum eucalyptorum infection

Eucalyptus species are widely spread over the world, being extensively planted and exploited by industries. Drought and pathogens are known to affect the establishment and productivity of Eucalyptus plantations worldwide. The aim of this work was to evaluate the pathogenicity of Neofusicoccum eucalyptorum in drought‐stressed and well‐watered E. globulus plants. The effect of a previous drought priming step and the role of water status at the time of inoculation were evaluated. Lesion length, plant growth and physiological parameters (relative water content, water potential, photosynthetic pigments and lipid peroxidation) were determined. The results indicate that water‐stressed plants were more susceptible to N. eucalyptorum than non‐stressed ones. However, this response was particularly relevant when the plants were inoculated while water limitation was already occurring. Moreover, drought‐primed plants were slightly more resistant to fungal infection than the non‐primed ones. This study reinforces the importance of exploring drought × disease interaction in Eucalyptus and the underlying physiological responses involved in plant performance.     

Damping-off of soybean in southern Brazil can be associated with different species of Globisporangium spp. and Pythium spp.

Damping-off of soybean is a common problem in some fields in southern Brazil. Frequently, Phytophthora sojae has been pointed out as the main causal agent. However, questions were raised as to whether other oomycetes could also be present. Hence, the objectives of this study were to isolate, identify, and determine the pathogenicity and aggressiveness of oomycetes isolated from soybean seedlings. Thirty-three isolates were obtained from 13 fields. Isolates were identified based on morphology and molecular methods. Nine species were recovered: Pythium conidiophorum (n = 14, 42.4%), Globisporangium rostratifingens (n = 7, 21.2%), G. irregulare (n = 2, 6.1%), G. spinosum (n = 1, 3.0%), G. ultimum var. sporangiiferum (n = 2, 6.1%), Pythium acanthicum (n = 2, 6.1%), P. deliense (n = 1, 3.0%), P. inflatum (n = 1, 3.0%), and P. torulosum (n = 3, 9.1%). Pathogenicity and aggressiveness were determined with two soybean cultivars (DM57i52 and NEO530) in a seed rot assay and a root rot assay. All isolates were pathogenic to soybean with variable levels of aggressiveness in both assays. The range of seed disease severity index varied from 7.5 to 100 for DM57i52 and from 23.1 to 100 for NEO530. The mean root disease severity index of the soybean cultivars ranged from 29.4 to 92.8. This is the first official report of G. irregulare, G. rostratifingens, G. spinosum, G. ultimum var. sporangiiferum, P. acanthicum, P. conidiophorum, P. deliense, P. inflatum, and P. torulosum causing damping-off of soybean in the state of Rio Grande do Sul in southern Brazil.     

Variability in Geminivirus Isolates Associated with Phaseolus spp. in Brazil

ABSTRACT Bean golden mosaic geminivirus (BGMV) is the single most devastating virus of common beans in the tropical and subtropical Americas and the Caribbean Basin. The BGMV from Brazil, named BGMV-BZ, is considered distinct from BGMV-PR isolates from Puerto Rico, Guatemala, and the Dominican Republic because of DNA sequence data, the ability to form pseudorecombinants, and mechanical transmissibility properties. In bean-growing areas of Brazil, samples were collected from beans, lima beans, and the weed Leonurus sibiricus displaying typical symptoms of infection by geminiviruses. Viral DNA fragments comprising part of the rep gene, the common region, and part of the cp gene were amplified by polymerase chain reaction, cloned, and sequenced. The bean samples had geminivirus with sequences nearly identical to that of BGMV-BZ collected in Goiania, state of Goiás, in 1986. The sample from lima bean contained a new species of geminivirus that induces symptoms similar to those induced by BGMV-BZ and was named lima bean golden mosaic virus (LBGMV-BR). While all sequences from bean samples clustered with BGMV-BZ, the sequence from the lima bean isolate stood alone. A mixed infection with abutilon mosaic geminivirus was also found in a single sample from the state of S?o Paulo. DNA sequence comparisons indicate that the virus isolate from L. sibiricus represents a new geminivirus species, designated here as leonurus mosaic virus.     

Yellow sigatoka epidemics caused by a panmictic population of Mycosphaerella musicola in Brazil

The genetic structure of Mycosphaerella musicola has never been investigated in Brazil to address epidemiologically related questions associated with yellow sigatoka. A total of 223 single‐conidium isolates from four regions (North, South, Zona da Mata and Triângulo Mineiro) of Minas Gerais State, Brazil, were used to assess the population genetic structure of M. musicola. Isolates were characterized regarding the frequency of the MAT1‐1‐1 or MAT1‐2‐1 idiomorphs and polymorphism at nine microsatellite loci. The mating‐type ratio in three of the four subpopulations was c. 1:1. A total of 87 alleles and 216 multilocus genotypes were identified. The overall population was in linkage equilibrium. Most (93.9%) genetic variation was detected within the subpopulations and there was weak differentiation between them. In total, eight genetic groups were detected and isolates of seven groups were present in all regions. The population of M. musicola in Minas Gerais seems to have high evolutionary potential: it is panmictic and both sexual reproduction and gene flow affect genetic variability. Strategies to avoid fungicide resistance should be enforced and breeding programmes need to consider quantitative resistance in the banana cultivars.     

Identification of Phytophthora spp. isolated from plants and soil samples on strawberry plantations in Poland

Journal of Plant Diseases and Protection - Crown and leather rot of strawberry caused by Phytophthora spp. are major soil diseases of cultivated strawberry...     

<Emphasis Type="Italic">Teratosphaeria (Mycosphaerella) nubilosa,</Emphasis> the causal agent of Mycosphaerella leaf disease (MLD), recently introduced into Uruguay

Mycosphaerella leaf disease on Eucalyptus is well known in Uruguay but none of the more serious Mycosphaerella spp. and Teratosphaeria spp. causing this disease have yet been found. In the autumn of 2007, more severe defoliation than has been known in the past and associated with symptoms resembling Mycosphaerella infections was observed on Eucalyptus globulus. Isolations and identifications based on morphology and DNA sequence comparisons showed that the causal agent of the defoliation is the well known and serious pathogen Teratosphaeria nubilosa (=Mycosphaerella nubilosa). This is the first record of the pathogen in South America. Using ten microsatellite loci previously developed for T. nubilosa, only one multilocus haplotype was found from 46 T. nubilosa collected isolates. Interestingly, this haplotype was the same as one previously found in Portugal and Spain. The results suggest that T. nubilosa has recently been introduced into Uruguay and that it most likely originated from the Iberian Peninsula where E. globulus is widely planted.