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.     

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.     

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.     

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.     

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.     

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.     

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.     

<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.     

Diversity and potential impact of Botryosphaeriaceae species associated with <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> plantations in Portugal

Eucalyptus globulus, a non-native species, is currently the most abundant forest species in Portugal. This economically important forest tree is exploited mainly for the production of pulp for the paper industry. The community of Botryosphaeriaceae species occurring on diseased and healthy E. globulus trees was studied on plantations throughout the country. Nine species from three different genera were identified, namely Botryosphaeria (B. dothidea), Diplodia (D. corticola and D. seriata) and Neofusicoccum (N. australe, N. algeriense, N. eucalyptorum, N. kwambonambiense, N. parvum and Neofusicoccum sp.). Of these, N. algeriense, D. corticola and D. seriata are reported for the first time on E. globulus, while N. algeriense, N. eucalyptorum and N. kwambonambiense correspond to first reports in Portugal. The genus Neofusicoccum was clearly dominant with N. australe and N. eucalyptorum being the most abundant species on both diseased and healthy trees. In artificial inoculation trials representative isolates from all nine species were shown to be pathogenic to E. globulus but there were marked differences in aggressiveness between them. Thus, D. corticola and N. kwambonambiense were the most aggressive while B. dothidea and D. seriata were the least aggressive of the species studied.     

Black Sigatoka leaf streaks of banana (Musa spp.) caused by Mycosphaerella fijiensis in Ethiopia

Journal of Plant Diseases and Protection - Sigatoka leaf streaks caused by Mycosphaerella spp. are the most destructive fungal diseases of bananas and plantains throughout the world. The disease is...     

Genetic Diversity in Colletotrichum gloeosporioides from Stylosanthes spp. at Centers of Origin and Utilization

ABSTRACT Using molecular markers, this work compares the genetic diversity in Colletotrichum gloeosporioides infecting species of the tropical forage legume Stylosanthes at the center of origin in Brazil and Colombia with that of Australia, China, and India, where Stylosanthes spp. have been introduced for commercial use. There was extensive diversity in the pathogen population from Brazil, Colombia, China, and India. The Australian pathogen population was least diverse probably due to its geographical isolation and effective quarantine. The extensive diversity in China and India means that threats from exotic pathogen races to Stylosanthes pastures can potentially come from countries outside the South American center of origin. In Brazil and India, both with native Stylosanthes populations, a high level of genetic differentiation in the pathogen population was associated with sites where native or naturalized host population was widely distributed. There was limited genetic diversity at germplasm evaluation sites, with a large proportion of isolates having identical haplotypes. This contrasts recent pathogenicity results for 78 of the Brazilian isolates that show hot spots of complex races are more common around research stations where host germplasm are tested, but few are found at sites containing wild host populations. For a pathogen in which the same races arise convergently from different genetic backgrounds, this study highlights the importance of using both virulence and selectively neutral markers to understand pathogen population structure.     

Potential use of Eucalyptus globulus essential oil against Phytophthora colocasiae the causal agent of taro leaf blight

This study was carried out to determine the antimicrobial activity of essential oil derived from leaves of eucalyptus (Eucalyptus globulus) against Phytophthora colocasiae, the causal agent of taro leaf blight (TLB). Essential oil was obtained by Clevenger-type water distillation. The major compounds in this essential oil were identified using gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC/MS). Antimicrobial activity of the essential oil was tested in vitro against mycelial growth, sporangia and zoospores germination of P. colocasiae. Additionally, in situ tests were conducted on detached healthy taro leaves discs and evaluating necrosis symptoms of TLB were assessed. Results of the chemical composition of the essential oil analysis showed that, 1.8-cineole (26.4 %), α-pinene (14.1 %) and p-cymene (10.2 %) are the most abundant compounds. In addition, four components could be identified in noticeable amounts (18.1 % in the total): regulator G1/G2 and its derivatives as well as flavesone, a regulator G3 derivative. G is a generic term for a family of 2,3-dioxabicyclo[4,4,0] decane system growth regulators. Here, G-regulators and derivatives are described for the first time in E. globulus. Antimicrobial activity of the essential oil was recorded with total inhibition of mycelia growth and sporangia germination at 0.625 mg/ml, while the complete inhibition of zoospores germination was recorded at 0.156 mg/ml. In situ results showed that essential oil completely inhibited the appearance of disease symptoms, necrosis development and sporulation at 3.5 mg/ml. These results demonstrated that, the essential oil of Eucalyptus globulus has high ability to inhibit the development of P. colocasiae, and might be used for controlling TLB.     

Genotypic Diversity of the Wheat Leaf Blotch Pathogen Mycosphaerella Graminicola (anamorph) Septoria Tritici in Germany

The population structure and genotypic diversity of Mycosphaerella graminicola from six natural field populations in Germany were studied with molecular markers. To reveal the potential effects of plant host resistance on the pathogen population, hierarchical samples were taken from susceptible and resistant cultivars. A total of 203 single spore isolates was subjected to molecular marker analysis using the amplified fragment length polymorphism technique (AFLP). Among the 203 isolates analyzed, 142 different multilocus haplotypes (MLH) were identified revealing a high degree of genotypic diversity of the M. graminicola population. On average, a F _ST value of 0.04 was found, indicating a low genetic differentiation with only 4% of the genetic variation between the local populations but leaving 96% of the genetic variation within the populations. According to the low F _ST value, a high migration rate of Nm 12 was found. The observed high within-population diversity, and the significant migration between populations, prevented genetic isolation and differentiation of putative geographically separated populations. Furthermore, plant host resistance had no obvious effect on the population structure and diversity of M. graminicola. Genotypic variability can be attributed to sexual recombination which appears to have a considerably larger influence on the population structure. Gene flow on this scale could have significant implications for plant breeding and fungicide spraying programmes.     

rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil

In this paper we present the first report of the occurrence of a binucleate Rhizoctonia spp. causing hypocotyl and root rot in kale in Brazil. Rhizoctonia spp. were isolated from kale (Brassica oleracea var. acephala) with symptoms of hypocotyl and root rot. The isolates, characterized as binucleate Rhizoctonia spp., did not show an anastomosis reaction with any of the binucleate Rhizoctonia spp. testers used. The pathogenicity of the isolates was tested under greenhouse conditions; all isolates were pathogenic and showed different symptom severities on kale. The ITS-5.8S rDNA sequences of kale isolates and 50 testers (25 binucleate Rhizoctonia spp. and 25 Rhizoctonia solani) were compared in order to characterize the genetic identity of Rhizoctonia spp. infecting kale. The kale isolates showed genetic identities ranging from 99.3 to 99.8% and were phylogenetically closely related to CAG 7 (AF354084), with identities of 98.5 and 98.7%. It is suggested that the binucleate Rhizoctonia spp. causing hypocotyl and root rot on kale Brazil comprises a new AG not yet described.     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

Characterization of Rhizoctonia Species Associated with Foliar Necrosis and Leaf Scorch of Clonally-propagated Eucalyptus in Brazil

The objective was to identify and characterize the causal agent of foliar necrosis and leaf scorch of Eucalyptus spp. in Brazil. Nineteen putative isolates of Rhizoctonia obtained from Eucalyptus plants during clonal propagation were compared with isolates from other hosts and with tester strains of anastomosis groups of Rhizoctonia solani. Features compared were morphological characteristics of anamorphs and teleomorphs, numbers of nuclei per cell in the vegetative hyphae, anastomosis of hyphae, and ability to produce necrotic lesions on cuttings and damping-off of E. grandis×E. urophylla hybrid seedlings. Rhizoctonia solani AG1 (AG1-IB like) was the most frequent causal agent isolated from Eucalyptus plants and cuttings with symptoms of leaf scorch and foliar necrosis respectively. These isolates were highly virulent on Eucalyptus cuttings and presented naturally epiphytic growth on Eucalyptus shoots. Binucleate isolates and isolates of R. solani AG4 were also virulent on cuttings and were most virulent on Eucalyptus seedlings causing pre- and post-emergence damping-off. Virulence on Eucalyptus cuttings and seedlings was not restricted to a single species or anastomosis group of Rhizoctonia.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.