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.     

Description and molecular phylogeny of <Emphasis Type="Italic">Ditylenchus gilanicus</Emphasis> n. sp. (Nematoda: Anguinidae) from northern forests of Iran

Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.     

Risk assessment for Puccinia psidii becoming established in South Africa

Puccinia psidii, the causal agent of myrtle rust, was first recorded from Latin America more than 100 years ago. It occurs on many native species of Myrtaceae in Latin America and also infects non‐native plantation‐grown Eucalyptus species in the region. The pathogen has gradually spread to new areas including Australia and most recently South Africa. The aim of this study was to consider the susceptibility of selected Eucalyptus genotypes, particularly those of interest to South African forestry, to infection by P. psidii. In addition, risk maps were compiled based on suitable climatic conditions and the occurrence of potential susceptible tree species. This made it possible to identify the season when P. psidii would be most likely to infect and to define the geographic areas where the rust disease would be most likely to establish in South Africa. As expected, variation in susceptibility was observed between eucalypt genotypes tested. Importantly, species commonly planted in South Africa show good potential for yielding disease‐tolerant material for future planting. Myrtle rust is predicted to be more common in spring and summer. Coastal areas, as well as areas in South Africa with subtropical climates, are more conducive to outbreaks of the pathogen.     

Evidence that Austropuccinia psidii may complete its sexual life cycle on Myrtaceae

The rust fungus Austropuccinia psidii has spread globally and naturalized in areas with naïve species of Myrtaceae. Previous studies have revealed multiple strains of A. psidii within South America and two strains outside of its native range. The rust spreads by windborne mitotic urediniospores, which are the dominant spore stage. Teliospores and basidiospores of A. psidii are also formed; however, the biological role of these stages in the life cycle is unknown. Experiments presented here tested whether basidiospores of A. psidii could infect Syzygium jambos. The sori produced by infection with basidiospores were screened with five microsatellite markers to confirm whether they were a product of recombination. The findings showed that basidiospores of A. psidii could cause infection on species of Myrtaceae and the resulting sori were a product of recombination. This has important implications for programmes that breed for resistance to this aggressive pathogen in commercial eucalypt forestry.     

Screening Corymbia populations for resistance to Puccinia psidii

The exotic rust pathogen Puccinia psidii is now widespread along the east coast of Australia from temperate Victoria to tropical far north Queensland, with a current host range exceeding 200 species from 37 myrtaceous genera. To determine the threat P. psidii poses to plantation and native eucalypts, artificial inoculation was used to screen germplasm of spotted gum (Corymbia spp.) for resistance to the biotype of P. psidii that has become established in Australia. The objective was to characterize resistance to P. psidii within the Corymbia species complex so that management strategies for the deployment of germplasm from existing breeding programmes of these spotted gum species could be developed. Symptom development initiated 7 days after inoculation, with resistant and susceptible seedlings identified within all species, provenances and families. Inter‐ and intraspecific variability in rust resistance was observed among spotted gum species. There was no apparent relationship between climatic conditions at the provenance origin and disease resistance. The heritability estimates for all assessments are moderate to high and indicate a significant level of additive genetic variance for rust resistance within the populations. The results of this study clearly identify potential to select for resistance at the family level within the tested populations. While the potential for P. psidii to detrimentally impact upon Corymbia in the nursery and in young plantations was demonstrated, estimations of the heritability of resistance suggest that efforts to enhance this trait through breeding have reasonable prospects for success.     

Glyphosate reduces urediniospore development and Puccinia psidii disease severity on Eucalyptus grandis

BACKGROUND: Recent studies have shown the effects of glyphosate drift on decreasing rust intensity on Eucalyptus grandis plants. However, the effects of the herbicide on Puccinia psidii initial development are unknown. In this study the systemic action of glyphosate on rust severity was evaluated on Eucalyptus plants maintained under greenhouse conditions. Urediniospore germination and apressorium formation on detached leaves and on water agar medium, previously treated with glyphosate, were also evaluated. RESULTS: Rust severity and the number of urediniospores per leaf area were significantly reduced with increasing glyphosate doses, even on branches not directly treated with the herbicide, indicating a systemic effect of glyphosate on pathogen development. Similarly, higher glyphosate doses also reduced germination and apressorium formation on detached Eucalyptus leaves, regardless of the direct application of the product on the leaf limb or on the petiole base. Puccinia psidii urediniospore germination in water agar medium also decreased with increasing herbicide doses. CONCLUSIONS: Reductions in germination and apressorium formation of P. psidii urediniospores with increasing glyphosate dose indicate that a lower severity and intensity of the disease may perhaps be due to blockage of the shikimic acid pathway in the fungal metabolic system. Copyright © 2011 Society of Chemical Industry     

Simple sequence repeat markers support the presence of a single genotype of Puccinia psidii in Australia

A non‐native rust of Myrtaceae was first detected in Australia in 2010, and was later identified as Puccinia psidii. The presence of many native species of Myrtaceae and a lack of understanding of genetic variability in P. psidii in Australia led to the current study. Low coverage genome sequencing of P. psidii suggested a genome size of c. 142 Mb. A set of 240 simple sequence repeat (SSR) primers was designed based on the genome sequence information generated. Seventeen isolates of P. psidii comprising 14 from Australia, two from Brazil and one from Hawaii were selected to study genetic variation in the pathogen. Out of 240 initially screened markers, 74% showed amplification among P. psidii isolates and 38% were polymorphic. Primers were fluorescently labelled and genotyping revealed three distinct genotypes among the isolates: one comprising Australian isolates and an isolate from Hawaii, and the second and third comprising two Brazilian isolates. Locus USYD_Pp151 produced a fourth genotype for the Hawaiian isolate of P. psidii. Markers revealed that all Australian isolates were genetically similar to the one from Hawaii. There was no genetic variation among the Australian isolates of P. psidii, supporting the hypothesis that only one genotype of P. psidii was introduced into Australia. The SSR markers developed in this study are highly specific to P. psidii and can be used confidently as a new profiling tool to monitor evolution of P. psidii in Australia and elsewhere.     

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.     

Eucalyptus rust caused by Puccinia psidii and the threat it poses to Australia1

Puccinia psidii, which causes the disease Eucalyptus rust, poses a threat to biodiversity in Australia and the Eucalyptus forest industry worldwide. It is native to South America and Central America and has spread to North America (Mexico, USA – Florida). In mid‐2005, the rust was reported in Hawaii, USA, which means it is now present in the Pacific region.     

Characterization of <Emphasis Type="Italic">Puccinia psidii</Emphasis> isolates in Hawaii using microsatellite DNA markers

We studied genetic variation in Puccinia psidii, a newly introduced rust fungus in Hawaii, using microsatellite markers. All 36 Hawaiian P. psidii isolates analyzed had the same genotype at 11 microsatellite loci, while three genotypes were identified among four isolates from Florida and 11 genotypes among 18 isolates from Brazil. One of the isolates from Florida had 95% similarity to the Hawaiian isolates. We conclude that the genetically homogeneous P. psidii isolates in Hawaii might have derived from the same strain originally introduced into the Islands and are closely related to one of the strains found in Florida.     

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.     

Projecting the suitability of global and local habitats for myrtle rust (Austropuccinia psidii) using model consensus

Myrtle rust (caused by Austropuccinia psidii) affects more than 500 known host species in the Myrtaceae family. Three different modelling approaches (CLIMEX, MaxEnt and Multi-Model Framework) were used to project the habitat suitability for myrtle rust at both global and local scales. Current data on the global occurrence of myrtle rust were collected from online literature and expert solicitation. Long-term averages of climate data (1960–1990) were sourced from WorldClim and CliMond websites. Recent reports of myrtle rust in New Zealand were used for validation of model outputs but not in model training and testing. The model outputs were combined into a consensus model to identify localities projected to be suitable for myrtle rust according to two or three models (hotspots). In addition to the locations where the pathogen is currently present, all models successfully projected independent occurrence data in New Zealand suitable for establishment of the pathogen. Climate suitability for the pathogen was primarily related to temperature followed by rainfall in MaxEnt and the CLIMEX model. The results confirmed the optimum temperature range of this pathogen in the literature (15–25 °C). Additional analysis of the precipitation variables indicated that excessive rain (more than 2000 mm in warmest quarter of the year) combined with high temperatures (>30 °C) constrain pathogen establishment. The results of the current study can be useful for countries such as New Zealand, China, South Africa and Singapore where the pathogen has not fully spread or established.     

Puccinia psidii in Queensland,Australia: disease symptoms,distribution and impact

Puccinia psidii has long been considered a significant threat to Australian plant industries and ecosystems. In April 2010, P. psidii was detected for the first time in Australia on the central coast of New South Wales (NSW). The fungus spread rapidly along the east coast and in December 2010 was found in Queensland (Qld) followed by Victoria a year later. Puccinia psidii was initially restricted to the southeastern part of Qld but spread as far north as Mossman. In Qld, 48 species of Myrtaceae are considered highly or extremely susceptible to the disease. The impact of P. psidii on individual trees and shrubs has ranged from minor leaf spots, foliage, stem and branch dieback to reduced fecundity. Tree death, as a result of repeated infection, has been recorded for Rhodomyrtus psidioides. Rust infection has also been recorded on flower buds, flowers and fruits of 28 host species. Morphological and molecular characteristics were used to confirm the identification of P. psidii from a range of Myrtaceae in Qld and compared with isolates from NSW and overseas. A reconstructed phylogeny based on the LSU and SSU regions of rDNA did not resolve the familial placement of P. psidii, but indicated that it does not belong to the Pucciniaceae. Uredo rangelii was found to be con‐specific with all isolates of P. psidii in morphology, ITS and LSU sequence data, and host range.     

Detecting myrtle rust (Austropuccinia psidii) on lemon myrtle trees using spectral signatures and machine learning

Hundreds of species in one of Australia's dominant plant families, the Myrtaceae, are at risk from the invasive pathogenic fungus Austropuccinia psidii. Since its arrival in Australia in 2010, native plant communities have been severely affected, with highly susceptible species likely to become extinct from recurring infections. While severe impact on Australian native and plantation forestry has been predicted, the lemon myrtle industry is already under threat. Commercial cultivars of lemon myrtle (Backhousia citriodora) are highly susceptible to A. psidii. Detecting and monitoring disease outbreaks is currently only possible by eye, which is costly and subject to human bias. This study aims at developing a proof‐of‐concept for automated, non‐biased classification of healthy (naïve), fungicide‐treated and diseased lemon myrtle trees by means of their spectral reflectance signatures. From a lemon myrtle plantation, spectral signatures of fungicide‐treated and untreated leaves were collected using a portable field spectrometer. A third class of spectra, from naïve lemon myrtle leaves that had not been exposed to A. psidii, was collected from a botanical garden. Reflectance spectra in their primary form and their first‐order derivatives were used to train a random forest classifier resulting in an overall accuracy of 78% (kappa = 0.68) for primary spectra and 95% (kappa = 0.92) for first‐order derivative‐transformed spectra. Thus, an optical sensor‐based discrimination, using spectral reflectance signatures of this as yet uninvestigated pathosystem, seems technically feasible. This study provides a foundation for the development of automated, sensor‐based detection and monitoring systems for myrtle rust.     

Symptom development and latent period of Austropuccinia psidii (myrtle rust) in relation to host species,temperature, and ontogenic resistance

Myrtle rust (Austropuccinia psidii) is an invasive species causing damage to Myrtaceae species in natural and managed ecosystems in many countries. To better understand myrtle rust epidemiology we studied latent period (LP) and ontogenic resistance in relation to temperature on three susceptible hosts (Metrosideros excelsa, Lophomyrtus bullata × L. obcordata and Syzygium jambos). The latent period curve was U-shaped, with latent development >0 from between 8 and 10 °C, depending on the host, to 32 °C. Optimum range was 22–28 °C with minimum LP of 5–7 days. Peak spore production occurred over about 2 weeks, starting about 1 week after the LP ended. Some spore production continued for 1–2 months. Comparison of the LP data with field temperatures indicated that the uredinial stage of A. psidii can overwinter in the latent phase in temperate areas of New Zealand and southern Australia and, therefore, uredinial or telial reinfection is not required during winter. The LP information was used to correct the LP function in a New Zealand myrtle rust climatic risk model. The transition of emergent leaf and stem tissues in susceptible Myrtaceae genotypes from susceptible to immune (ontogenic resistance) was characterized in terms of uredinium density and LP. Onset of ontogenic resistance was closely linked to the degree of leaf expansion, with fully expanded leaves being immune to infection. Because ontogenic resistance restricts infection to periods when growth flushes occur, understanding it is crucial for explaining the seasonality of myrtle rust development in the natural environment.     

Bidirectional colonization and biofilm formation by Erwinia psidii in eucalypt plants

Dieback and wilt caused by Erwinia psidii is an emerging disease that has been causing considerable damage in eucalypt plantations. Because it is a recently emerged disease, several aspects of the bacterial interaction with its host still remain to be elucidated. In this work, we studied the E. psidii colonization and biofilm formation in eucalypt tissues by specific detection using PCR and scanning electron microscopy (SEM). The results indicate that the bacterium is able to translocate in stem tissue mainly acropetally, although movement in the basipetal direction was also observed to a lesser extent, always through the xylem. No colonization of phloem tissues was observed. In addition to colonizing the xylem, E. psidii colonized the parenchymatous tissue. The bacterium formed cell aggregates enveloped by fibrillar material that evolved into complex, well-structured biofilms in stem and leaf tissues. In contrast, no biofilm formation was observed on abiotic surfaces. These observations suggest that biofilm formation plays an important role in the elicitation of dieback and wilt symptoms caused by E. psidii on eucalypt plants. This study not only shows ultrastructural aspects of the E. psidii communities but also tissue damage in eucalypt plants that was associated with the presence of bacterial aggregates and formation of tyloses.     

A long‐term study of burning effects on a plant pathogen in tallgrass prairie

Tallgrass prairie species have evolved with regular exposure to fire. However, burning has been used as a management tool for reducing plant disease in agricultural systems, posing the question of how plant pathogens of tallgrass prairie would be affected by burning. The rust fungus Puccinia dioicae, infecting Erigeron strigosus (Asteraceae), was studied for 8 years in long‐term experiments to evaluate the effects of burning in native tallgrass prairie. This experiment also allowed evaluation of the effects of nutrient additions, although E. strigosus was rare in the plots with added nutrients in most years. Burning reduced rust severity in most years, but effects from additions of nutrients were rarely observed. There was high interannual variation in rust severity within a location, suggesting that weather may be the most important of these three abiotic factors in determining infection. An analysis of weather variables associated with disease severity found that solar radiation in the month prior to sampling was associated with severity in unburned plots; temperature approximately 2 months prior to sampling was also associated with severity in burned plots. High interannual variation also suggests that the effects of this pathogen on its host would be sporadic and difficult to study in short‐term experiments.     

White rust of <Emphasis Type="Italic">Ipomoea</Emphasis> caused by <Emphasis Type="Italic">Albugo ipomoeae-panduratae</Emphasis> and <Emphasis Type="Italic">A. ipomoeae-hardwickii</Emphasis> and their host specificity

In some areas of Japan, yellow spots with white pustules on leaves, stems, petioles, peduncles and calyces were found on Ipomoea nil, I. triloba, I. lacunosa and I. hederacea var. integriuscula. We demonstrated that the diseases on I. nil, I. triloba and I. lacunosa were caused by host-specific strains of Albugo ipomoeae-panduratae and defined three forma speciales of the fungus, respectively, for the three Ipomoea species: “f. sp. nile”, “f. sp. trilobae” and “f. sp. lacunosae”. Because the diseases were new to Japan, we coined the Japanese name “shirosabi-byo”, which means white rust. We also showed that the disease on I. hederacea var. integriuscula was caused by A. ipomoeae-hardwickii. We named this new disease “white rust (shirosabi-byo in Japanese)”.     

Choanephora rot of floral tops of <Emphasis Type="Italic">Hyoscyamus muticus</Emphasis> caused by <Emphasis Type="Italic">Choanephora cucurbitarum</Emphasis>

Floral rot of Egyptian henbane (Hyoscyamus muticus L.) was found on potted plants in a greenhouse in Yamaguchi city, Japan, in the late summer of 2008 and 2009. The symptoms were identical to those of rots caused by Choanephora species. The pathogen was isolated and identified as C. cucurbitarum (Berkeley and Ravenel) Thaxter. This new disease was named Choanephora rot (Kougai-kabi-byo) of Egyptian henbane.     

Characterization of Erwinia sp. causing black rot of papaya (Carica papaya) first recorded in Okinawa Main Island,Japan

Since 2002, papaya black rot has been spreading over several islands of Okinawa Prefecture, Japan. To devise a prevention strategy for the disease, microbiological research on the pathogen was conducted. Twelve strains were isolated from papaya infected with black rot showing symptoms such as water-soaked lesions on stems and petioles, black spots on fruits, and rotted leaves turning yellow with necrotic spots. Through Koch's postulates, we confirmed that the isolated strains caused papaya black rot. Bacteriological assays showed that the strains have characteristics different from the type strains of Erwinia mallotivora, E. papayae, and E. psidii. Moreover, 16S rDNA sequence similarity searches showed that the isolated strains had less than 98.6% similarity with type strains. Additionally, phylogenetic analysis of 16S rDNA sequences suggested that the isolated strains were possibly a novel species belonging to the genus Erwinia, as the strains formed an independent cluster and had low sequence similarity with the type strains. Earlier studies indicated that papaya black rot is caused by E. cypripedii. Therefore, we propose to add the Erwinia sp. isolated in this study to the list of papaya black rot pathogens.