Genetic diversity of the myrtle rust pathogen (Austropuccinia psidii) in the Americas and Hawaii: Global implications for invasive threat assessments

Since the myrtle rust pathogen (Austropuccinia psidii) was first reported (as Puccinia psidii) in Brazil on guava (Psidium guajava) in 1884, it has been found infecting diverse myrtaceous species. Because A. psidii has recently spread rapidly worldwide with an extensive host range, genetic and genotypic diversities were evaluated within and among A. psidii populations in its putative native range and other areas of myrtle rust emergence in the Americas and Hawaii. Microsatellite markers revealed several unique multilocus genotypes (MLGs), which grouped isolates into nine distinct genetic clusters [C1–C9 comprising C1: from diverse hosts from Costa Rica, Jamaica, Mexico, Puerto Rico, and USA‐Hawaii, and USA‐California; C2: from eucalypts (Eucalyptus spp.) in Brazil/Uruguay and rose apple (Syzygium jambos) in Brazil; C3: from eucalypts in Brazil; C4: from diverse hosts in USA‐Florida; C5: from Java plum (Syzygium cumini) in Brazil; C6: from guava and Brazilian guava (Psidium guineense) in Brazil; C7: from pitanga (Eugenia uniflora) in Brazil; C8: from allspice (Pimenta dioica) in Jamaica and sweet flower (Myrrhinium atropurpureum) in Uruguay; C9: from jabuticaba (Myrciaria cauliflora) in Brazil]. The C1 cluster, which included a single MLG infecting diverse host in many geographic regions, and the closely related C4 cluster are considered as a “Pandemic biotype,” associated with myrtle rust emergence in Central America, the Caribbean, USA‐Florida, USA‐Hawaii, Australia, China‐Hainan, New Caledonia, Indonesia and Colombia. Based on 19 bioclimatic variables and documented occurrences of A. psidii contrasted with reduced sets of specific genetic clusters (subnetworks, considered as biotypes), maximum entropy bioclimatic modelling was used to predict geographic locations with suitable climate for A. psidii which are at risk from invasion. The genetic diversity of A. psidii throughout the Americas and Hawaii demonstrates the importance of recognizing biotypes when assessing the invasive threats posed by A. psidii around the globe.     

The risk to Myrtaceae of Austropuccinia psidii,myrtle rust,in Mexico

Austropuccinia psidii is a biotrophic rust fungus that affects species from the Myrtaceae family. In Mexico, Myrtaceae is widely distributed in temperate, tropical and semi‐arid ecosystems, and includes 20 genera and 192 endemic and exotic species. Austropuccinia psidii has been present in Mexico for the last four decades; however, little is known about the distribution of this rust or the vulnerability of native and exotic Myrtaceae to infection. In this study, we used global occurrence records for the pandemic biotype of myrtle rust to model its current and future suitable habitat using a species distribution model, Maxent. We identified regions that are highly suitable for myrtle rust establishment, now and in the future (2050). Additionally, we identified the Myrtaceae species known to be susceptible to rust infection and that are currently distributed in areas with high rust habitat suitability. Thirty‐six susceptible plant species and 142 untested species are distributed within areas of suitable rust habitat and are considered potentially at risk of rust infection. Current suitable habitat is mainly restricted to the east coast of Mexico, with Veracruz, Puebla, Chiapas, Tabasco and Oaxaca being the most vulnerable regions to the rust under current and future climates. We encourage monitoring within these regions by surveying locations where the rust occurs and within areas with high suitable habitat to determine the threat to native ecosystems and industries reliant on Myrtaceae. We also recommend screening to test the susceptibility of Myrtaceae species with no known susceptibility rating.     

Host range of Erwinia psidii and genetic resistance of Eucalyptus and Corymbia species to this pathogen

Dieback caused by Erwinia psidii is currently one of the most important emerging diseases in eucalypt plantations in Brazil. However, little is known in terms of the host range of this pathogen or the potential sources of resistance against the disease it causes. In this study, we inoculated plants of species from nine families to gain insight into the host range of E. psidii. Plants of all inoculated species of Myrtaceae except Acca sellowiana exhibited disease symptoms and therefore represent potential hosts for the pathogen under natural conditions. In addition, the response of four Corymbia species, 29 Eucalyptus species and three interspecific Eucalyptus hybrids to inoculation with E. psidii was evaluated. All Corymbia henryi, Corymbia maculata, Eucalyptus thozetiana, Eucalyptus cloeziana, Eucalyptus viminalis, Eucalyptus dalrympleana and Eucalyptus pilularis plants were highly resistant to the pathogen, whereas differential disease resistance was observed in the other species. This study provides important information on sources of resistance to Erwinia psidii with potential use in the development of clones with enhanced resistance in eucalypt species of economic importance.     

First report of Puccinia psidii on Corymbia citriodora and Eucalyptus in Colombia

A rust disease was observed during routine disease surveys of Eucalypt species in Colombia. The cause of the disease was identified, using morphology and DNA sequence data, as the myrtle rust pathogen, Puccinia psidii. We evaluated the susceptibility of Eucalyptus grandis and the hybrid Eucalyptus urograndis to P. psidii. This is the first report of this pathogen on Corymbia citriodora and Eucalyptus species in Colombia.     

Wind dispersal of Puccinia psidii urediniospores and progress of eucalypt rust

To identify and implement Puccinia psidii control strategies, it is essential to understand the role of environmental factors on rust‐disease development and spread among eucalypt (Eucalyptus spp.) plantations. In this study, we evaluated the wind dispersal of P. psidii urediniospores and the progress of eucalypt rust in a field trial in Brazil. Urediniospores of P. psidii were trapped in a Burkard^® spore trap from July 2004 to June 2005. To evaluate the progress of eucalypt rust, plots were established in March 2002 using a clonal hedge scheme. The incidence of both branches and leaves with rust was assessed weekly from February 2003 to October 2005. Disease progress was studied using time‐series analysis. Urediniospores were trapped on 77% of the days sampled. The highest average urediniospore concentration was detected from July to November, and most of the urediniospores (58%) were trapped at night. Urediniospore concentration was negatively correlated with rainfall, light intensity, minimum, average and maximum temperatures and wind speed, whereas urediniospore concentration was positively correlated with leaf‐wetness duration and relative humidity. From December 2004 to June 2005, average urediniospore concentration was 0.22 spores m^?3 air h^?1 and had no correlation with meteorological data. The highest average urediniospore concentration was associated with a combination of low average temperature, low light intensity, low wind speed, high relative humidity and high leaf wetness, which reflect conditions observed at night. The disease incidence was positively correlated with the urediniospores trapped at 12 days prior to disease assessment. According to the models obtained by the time series, a seasonal yearly effect was found on rust progress. Using our models, we were also able to forecast disease incidence up to 3 months after the last field assessment.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

Comparing Chilean and Swedish rust populations: possible impact of long‐distance transport of Melampsora rust

As willow (Salix) cultivation grows globally, worldwide monitoring of pathotypes for Melampsora rust, the most serious disease in willow, becomes increasingly important. We compared the pathotype composition of the Melampsora rust population in Chile with that in Sweden and assessed inocula exchange for the rust between the continents. For pathotyping rust isolates, a willow differential was used (a standardized set of willow test clones) to obtain clone‐virulence patterns. These patterns were used to identify virulence components involved in determining which willow clones a given individual pathogen isolate (genotype) can successfully attack. Each virulence component detected earlier in Sweden's rust population was present in Chile's rust population. Using isoenzyme analysis to classify willow species in Chile, a low genetic variation of Salix viminalis (an introduced willow species) was found, compared with the endemic species Salix humboldtiana. Comparison of rust populations for the two countries supports the present hypothesis that intercontinental inocula exchange can be a significant determinant of local pathotype structure, and consequently can be important for willow‐resistance breeding.     

A new species of Melampsora rust on Salix elbursensis from Iran

A rust fungus was found causing stem cankers on 1‐ to 5‐year‐old stems of Salix elbursensis in the north west of Iran. The rust also forms uredinia on leaves and flowers of the host willow. Light and scanning electron microscopy revealed that the new rust is morphologically distinct from several Melampsora species occurring on the willows taxonomically close to S. elbursensis, but indistinguishable from Melampsora larici‐epitea. Examination of the internal transcribed spacer (ITS) region of the ribosomal DNA suggested that the rust fungus is phylogenetically close to Melampsora allii‐populina and Melampsora pruinosae on Populus spp. Based on both the morphological characteristics and the ITS sequence data, the rust is described as a new species –Melampsora iranica sp. nov.     

Pedicularis and Castilleja are natural hosts of Cronartium ribicola in North America: a first report

White pine blister rust disease, caused by the introduced pathogen Cronartium ribicola, has severely disrupted five‐needled pine ecosystems in North America. A 100‐year effort to manage this disease was predicated in part on the premise that the pathogen utilizes only species of Ribes (Grossulariaceae) as alternate hosts on this continent. The current study presents the first conclusive demonstration that some species in the family Orobanchaceae (Pedicularis racemosa and Castilleja miniata) are functioning as alternate hosts in a natural ecosystem of North America. This finding has implications for improving our understanding of epidemiology, pathogen adaptation and host–pathogen interactions within white pine blister rust.     

Standard area diagram set to assess rust severity on eucalyptus leaves

This study aimed to develop and validate a standard area diagram set (SADs) to help assess rust severity caused by Austropuccinia psidii on eucalyptus leaves. The SADs has eight colour images of leaves with severity values ranging from 0.97% to 37.3%. The SADs were validated by 16 raters, who assessed the same set of 50 images twice: the first without the SADs and the second using it as an aid. Accuracy and precision of the estimates were determined based on Lin's concordance correlation coefficient, and the inter‐rater reliability was measured using the coefficient of determination (R²) and intraclass correlation. The estimates of severity of eucalyptus rust were more accurate, precise and reliable using SADs. These results demonstrate the value of SADs for several activities, including epidemiological studies, treatments comparison, disease monitoring, decision‐making in eucalyptus rust management measures, and in breeding programs.     

Phylogeography and host range of Armillaria gallica in riparian forests of the northern Great Plains,USA

Root disease pathogens, including Armillaria, are a leading cause of growth loss and tree mortality in forest ecosystems of North America. Armillaria spp. have a wide host range and can cause significant reductions in tree growth that may lead to mortality. DNA sequence comparisons and phylogenetic studies have allowed a better understanding of Armillaria spp. taxonomic diversity. Genetic sequencing has facilitated the mapping of species distributions and host associations, providing insights into Armillaria ecology. These studies can help to inform forest management and are essential in the development of disease risk maps, leading to more effective management strategies for Armillaria root disease. Armillaria surveys were conducted on publicly owned lands in North Dakota, South Dakota, and Nebraska, U.S.A. Surveyed stands consisted of riparian forests ≥0.4 hectares in area. Armillaria was found at 78 of 101 sites. A total of 57 Armillaria isolates—associated with 12 host tree species—were used for DNA sequencing of the translation elongation factor‐1 alpha (tef1) gene. Armillaria gallica was the only species identified within the study sites. Results suggest that A. gallica is a common root pathogen of hardwood trees in riparian forests of the northern Great Plains with a wider host range and geographic distribution than previously recognized.     

Wilt and die‐back of Eucalyptus spp. caused by Erwinia psidii in Brazil

A new disease of unknown bacterial aetiology has been observed in eucalyptus stands since 2009. It is characterized by die‐back, wilting and lesions on the branches, petiole and midrib in association with macroscopic and microscopic bacterial ooze. To date, this disease has been observed in stands of clonal Eucalyptus saligna, E. grandis and E. urophylla x E. grandis hybrids and in E. dunnii seedling plantations in the states of São Paulo, Rio Grande do Sul and Mato Grosso do Sul. Considering the economic importance of eucalyptus plantations and the potential losses caused by this disease, this study aimed to identify and characterize the causal agent. Thirty‐four strains were obtained from infected plants, which were collected in the field from four locations. The inoculation of detached leaves and intact rooted cuttings supported pathogenicity in eucalyptus. The phylogenetic analysis of four housekeeping genes (16S rDNA, gapA, recA and rpoB) as well as biochemical tests confirmed the identity of strains belonging to the species Erwinia psidii. This is the first report of E. psidii as the cause of wilt and die‐back in Eucalyptus spp. in Brazil.     

Climatic favourability zones for Eucalyptus rust in Brazil

Brazilian forest‐based industries are supported by more than 5.5 million hectares of Eucalyptus growing under different climatic conditions with different degrees of favourability for rust Puccinia psidii, including both traditional and expanding areas, where such disease is of major concern for the industry, foresters and scientists. The main objective of this study was to define favourable climatic zones for Eucalyptus rust in Brazil with the following aims: (i) to develop a spatial method for estimating the mean night‐time temperature (T_ng); (ii) to assess and validate a Eucalyptus rust model; and (iii) to map Eucalyptus rust favourability zones in Brazil based on the proposed model. A straightforward method, based only on latitude, day of the year, maximum and minimum air temperatures, was developed to estimate T_ng, which is a key variable for a proper application of Ruiz rust model. Based on 37 field experiments with natural rust occurrence, it was observed that climatic conditions are determining factors for disease severity. Significant correlations between disease severity observed in the field and climatic conditions were found: a negative correlation with temperature (r = .50, p < .01) and a positive correlation with relative humidity (r = .89, p < .0001). A significant correlation (r = .81, p < .0001) between the normalized infection index, produced from Ruiz model, and the average rust score was also observed. Once the model was validated under field conditions, it was applied using historical average data of air temperature and leaf wetness duration to obtain monthly Eucalyptus rust favourability maps for the whole country. These final maps show that favourable climatic zones for Eucalyptus rust are extremely dynamic, with high temporal and spatial variability in Brazil and that climatic conditions should be considered for expansion of Eucalyptus to new areas, in breeding programmes, and for defining the most suitable seasons for forest establishment in each climate of the country. These results provide forestry managers with practical tools to reduce uncertainty about the expected severity of Eucalyptus rust in Brazil.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

Methods of inoculation and evaluation of Erwinia psidii in eucalypt

The dieback and wilting caused by Erwinia psidii are emerging eucalypt diseases that have been observed since 2014 in the south and central‐south regions of Brazil. Field observations have shown variability in disease severity resistance among Eucalyptus spp. clones and species. It is hypothesized that this variability is due to genetic resistance. To confirm this hypothesis, inoculations in genetically distinct eucalypt plants are necessary. However, lack of an inoculation method and disease assessment makes difficult to select resistant genotypes for use in commercial plantations or genetic breeding programmes. Three inoculation methods were tested on eight clones of Eucalyptus spp. Among them, inoculum deposition with bacteria‐impregnated toothpick on the axillary buds was the simplest and most effective, capable to reproduce the disease symptoms observed under conditions of natural infection. We also developed a rating scale for disease assessment. Among eight clones tested, only Clone 1 (Eucalyptus saligna) and Clone 2 (Eucalyptus urophylla) were resistant.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

A rapid and holistic approach to screen susceptibility of Prunus species to Armillaria root rot

We describe a holistic in vitro technique for inoculating roots of Prunus species with Armillaria solidipes, which is faster and more successful than previous methods. This method allows associated active and passive host defences to be assessed. Sterile root segments of three Prunus spp. were placed next to or on top of 14‐day‐old cultures of A. solidipes. At 21 day, the success of fungal penetration and length of the fungal colonization, and host responses were evaluated. Also, an anti‐Armillaria and anti‐Cladosporium activity detection assays were conducted by utilizing the root periderm, the first tissue that needs to be penetrated by fungus. These methods revealed the variation in the relative tolerance of three Prunus spp. to A. solidipes. The overall success of fungal colonization in the wounded and intact root, host defence, and antifungal activity significantly differed among three Prunus spp. Results indicate that this in vitro method can be used as a preliminary step in screening tree species to Armillaria spp.     

Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

Mechanisms and metabolomics of the host–pathogen interactions between Chestnut (Castanea species) and Chestnut blight (Cryphonectria parasitica)

Chestnut blight is a stem‐girdling disease of Castanea caused by the fungal pathogen Cryphonectria parasitica. Chestnut blight affects all Castanea species to some degree. In Asian species, chestnut blight is a commercially relevant disease which primarily affects nut production. In American and European species, chestnut blight has caused significant declines in wild populations and continues to negatively affect nut production in the European chestnut (C. sativa). Despite the profound effect of this disease in the Castanea genus, very little is known concerning the factors involved in the host–pathogen interaction between C. parasitica and its Castanea hosts. This review summarizes information on known mechanisms and metabolites involved in the host–pathogen interaction and contributes original information on the pathogen in relation to susceptible and putatively resistant genotypes with a view to furthering research that will promote a better understanding of this devastating disease and enable its control.     

Castilleja and Pedicularis confirmed as telial hosts for Cronartium ribicola in whitebark pine ecosystems of Oregon and Washington

The primary objective of this research was to determine whether native species of Castilleja and Pedicularis are naturally infected by Cronartium ribicola in whitebark pine ecosystems of the Oregon and Washington Cascade Range, USA. Secondary objectives were to monitor the phenology of aecial and telial hosts to determine whether there is sufficient time for C. ribicola to complete its life cycle within high‐elevation stands and to evaluate the variety of susceptible native host species within these genera through field and growth chamber inoculation. These objectives were approached through fieldwork in 2008 and 2009 in whitebark pine ecosystems at Mt. Rainier, Mt. Adams, Mt. Hood, Mt. Bachelor, Tumalo Mtn. and Crater Lake. Forty‐nine observational study plots were established and monitored. Natural C. ribicola infection was detected on 84 Pedicularis racemosa plants and five Castilleja plants (C. applegatei, C. miniata and C. parviflora). Field observation provided evidence that there is sufficient time for C. ribicola to complete its life cycle on hosts within high‐elevation whitebark pine stands. In 2009, 18 field inoculation plots were established at Mt. Rainier and Crater Lake. Field inoculation confirmed the susceptibility of two additional species within these genera, C. arachnoidea and P. bracteosa. All four Castilleja species inoculated in the growth chamber developed infection, with an overall infection incidence of 62% (167 out of 270 plants). The identity of the rust species on field specimens as C. ribicola was verified through PCR and sequencing of the ITS1‐5.8S‐ITS2 region of DNA. Improved understanding of the role of these newly recognized hosts in white pine blister rust epidemiology should be used to prioritize sites for the restoration of ecologically valuable whitebark pine.