Increased atmospheric carbon dioxide concentration: effects on eucalypt rust (Puccinia psidii), C:N ratio and essential oils in eucalypt clonal plantlets

Evaluation of impacts of high CO₂ atmospheric concentration is strategically important for the development of adaptation measures and sustainable crop management. The objective of this study was to evaluate the effects of increased atmospheric CO₂ concentration on eucalypt rust (Puccinia psidii), C:N ratio, yield and chemical composition of essential oils and growth of eucalypt clonal plantlets. Two clones with different levels of rust resistance were studied: a Eucalyptus urophylla × E. camaldulensis hybrid (VM 01) and an E. urophylla (clone MN 463). The experiments were performed in open‐top chambers (OTCs) with CO₂ mean concentrations (μmol mol^?1) of 399 (unenclosed control), 412 (OTCs with ambient CO₂ concentration) and 508 (OTCs with high CO₂ concentration) and in closed chambers (CCs) with CO₂ mean concentrations of 390, 405, 520 and 700. Increased atmospheric CO₂ concentrations resulted in a decrease in rust pustules per leaf, uredinia per leaf area, spores per uredinia and area under the disease progress curve in VM 01 (hybrid) clonal plantlets. The disease did not occur in MN 463 clonal plantlets, which demonstrated that high CO₂ concentrations did not change the level of rust resistance. Plant growth of the two clones was stimulated by up to 23% in height and 26% in stem diameter in OTCs and by 18% for both clones in CCs. An increased C:N ratio in leaves, stems and roots was observed only for the VM 01 clonal plantlets. Essential oils produced by VM 01 (2.8 g 100 g^?1) and MN 463 (1.4 g 100 g^?1), as well as the major essential oil compounds (80% 1.8‐cineole for VM 01; 50% 1.8‐cineole and 32% α‐pinene for MN 463), were not altered. In this study, increased concentrations of atmospheric CO₂ favourably impacted eucalypt growth and reduced rust severity, while not influencing the production of essential oils.     

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.     

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.     

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.     

Impact of Austropuccinia psidii in New Caledonia,a biodiversity hotspot

Myrtle rust, caused by the fungus Austropuccinia psidii, infects a wide range of host species within the Myrtaceae family worldwide. Since its first report in 2013 from New Caledonia, the extent of the host range, geographical distribution and genetic diversity of A. psidii in this territory have been unknown. This study reports 67 new host species distributed in 13 different Myrtaceae genera, including five new genera. The pathogen was found in various types of plant communities, where Myrtaceae species are the dominant or codominant species, as well as in several nurseries. It is now considered a significant threat to the biodiversity of ecosystems and the Myrtaceae‐related economy. A 3‐year disease monitoring trial with 35 cultivated Myrtaceae species showed variations in disease severity at the species and individual scales. This suggests that some level of genetic resistance to myrtle rust may possibly exist among the endemic Myrtaceae of New Caledonia. A population genetic investigation revealed that only one single genotype of A. psidii occurs across its entire host range in New Caledonia. Therefore, it is essential to carry out population genetic surveillance to identify the appearance of mutations or the introduction of potentially more virulent genotypes of A. psidii. The outcomes from this study will assist with the ongoing management of the disease in New Caledonia.     

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.     

Screening of Corymbia and Eucalyptus species for resistance to Calonectria pteridis leaf blight

Calonectria leaf blight (CLB), caused by Calonectria pteridis, is a major foliar disease of eucalypt plantations in warm and high rainfall regions in Brazil. The use of resistant genotypes is the best method for disease control in the field, so identification of sources of resistance is strategic for the long‐term genetic breeding programmes of eucalypt. In this study, resistance of 13 species of Eucalyptus and 3 species of Corymbia to CLB was evaluated by spray inoculation of a spore suspension (1 × 10⁴ conidia ml^?1) of the pathogen, under controlled conditions. Eucalyptus brassiana, E. saligna, E. scias and E. agglomerata were the most resistant, while E. robusta, E. urophylla, E. camaldulensis, E. longirostrata and C. toreliana were moderately resistant and E. cloeziana, E. pellita, E. tereticornis, E. pilularis, C. maculata, E. grandis, E. dunnii and C. citriodora were the most susceptible. The broad inter and intraspecific variability of the species tested demonstrates the potential for introgression of resistance genes into valuable genotypes as a strategy for the breeding programmes and commercial plantation of eucalypt.     

Effect of Betula pendula clone and leaf age on Melampsoridium betulinum rust infection in a field trial

Ten 5‐year‐old Betula pendula clones were studied for their rust resistance in the field. The trees were treated by inoculating 10 leaves on a shoot with Melampsoridium betulinum urediniospore suspension or spraying the control leaves with water. The birch clones differed significantly in their resistance to M. betulinum leaf rust fungus and the clones also varied in their responses to the local rust strain and the inoculated rust strains. However, natural rust infections and inoculation treatment were positively correlated. The older leaves had fewer infections than the younger ones on the tip of the shoot in the control trees, but in the inoculation treatment no significant correlation was found between the leaf ages and rust infection. The factors behind the different leaf susceptibilities are discussed.     

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.     

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.     

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.     

Pre-penetration behaviour of the urediniospores of birch rust,Melampsoridium betulinum,on greenhouse-grown and microcultured leaves of Betula spp.

In two different experiments the urediniospores of Melampsoridium betulinum from Betula pendula and Betula pubescens germinated both on mature leaves of greenhouse-grown plants and on sterile in vitro leaves of micropropagated plantlets, which were cloned from the same B. pendula and B. pubescens genotypes. The urediniospores and germ tubes were more easily detached from the leaf surfaces of in vitro leaves. If germination took place on a leaf vein, the growth continued across the veinal ridges; otherwise, no determined growth towards the stomata could be observed with either of the leaf types studied. In both experiments on the in vitro leaves of B. pubescens clone V5944, the germ tubes of the rust isolate from B. pendula mislocated appressoria significantly more often than the germ tubes of the rust isolate from B. pubescens. On the mature leaves of B. pendula clone E4214 and B. pubescens clone V5940 there were also significant differences in appressorial locations between the two rust isolates but the clonal responses were inconsistent. The results of the inoculations suggest that the incompatibility of the rust isolates from B. pendula on the leaves of B. pubescens is not related to the significantly higher ratio of failures in locating appressoria in this host-rust combination.     

Screening of clones of Betula pendula and B. pubescens against two forms of Melampsoridium betulinum leaf rust fungus

Leaf discs from seedlings of six clones of B. pendula Roth and two clones of B. pubescens Ehrh. were inoculated with two urediniospore isolates of M. betulinum Kleb. The field collection iso-lates were obtained from B. pendula and B. pubescens growing in the field. The B. pendula rust was more specialized than the B. pubescens rust. The clones of B. pubescens showed partial resis-tance against the B. pendula rust, while the B. pubescens isolate was compatible to both birch species. Some interclonal variation was also found in both birch species. The results support an earlier suggestion by Klebahn that M. betulinum has two formae speciales.     

Protocol for screening rust disease resistance in Dalbergia sissoo

A protocol has been developed for inoculation of rust disease in Dalbergia sissoo using urediniospores of Maravilia achroa. This is the first time that disease symptoms caused by M. achroa were artificially produced. The method has been successfully employed for screening 15 clones of D. sissoo against rust disease. Clone S‐167 was the most resistant remaining free from infection. Clones S‐57, S‐106 and S‐124 were also resistant. Clones S‐19, S‐24 and S‐89 were susceptible.     

Effect of Cladosporium aecidiicola Thum. on the viability of urediniospores of Melampsora medusae Thum. in storage

The viability of urediniospores of Melampsora medusae decreased markedly when stored in mixture with the conidia of Cladosporium aecidiicola. The rate of decrease was more rapid in samples stored at 16°C with high concentration of Cladosporium (ratio of urediniospores to conidia 10:1 by weight) than at ?12°C and low concentration (20:1 by weight).     

Distribution of the larch brown rust caused byTriphragmiopsis laricinum (Uredinales) in Russian Far East

The larch brown rust caused byTriphragmiopsis laricinum was found onLarix cajandri in Primorsky Territory, Russia. This was a new host and locality record for the pathogen. Preliminary survey of the disease incidence in several localities in East and Northeast Russia and Yakutia-Sakha Republic indicated the restricted distribution of the rust in a few localities in Primorsky Territory. As previously reported in China,T. laricinum was proved to form uredinia in nature and to be able to propagate vegetatively by urediniospores. This suggests the fungus could become a devastating pathogen threatening a large-scale larch plantation in Far East of Russia. Field observations and the failure of basidiospore inoculation onto larch needles suggest the heteroecious life cycle ofT. laricinum.     

Rust resistance in Salix induced by inoculations with avirulent and virulent isolates of Melampsora larici‐epitea

Six Salix clones and four Melampsora larici‐epitea isolates were used in two leaf‐disc inoculation experiments to determine whether disease severity was affected by the presence of both virulent and avirulent rust genotypes. In the first experiment, an equal amount of urediniospores of a virulent isolate and an avirulent isolate was applied simultaneously using four levels of spore suspension. In the second, the willows were inoculated with one isolate first and then, after 3 days, with another using two spore concentrations. In the first experiment, overall rust spore production was reduced by 48.6% in inoculations with mixed inocula compared with that in the inoculations with single virulent isolates. In the second experiment, 20 of the 36 combinations involving pre‐inoculations with avirulent isolates showed significant reduction in spore production. The suppressive effects on rust sporulation were more obvious at the higher inoculum densities and on the clones S. × calodendron and S. × mollissima‘Q83’.     

Field ready: Development of a rapid LAMP-based colorimetric assay for the causal agent of white pine blister rust,Cronartium ribicola

The invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five-needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non-Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species-specific real-time PCR assay to develop a field-ready loop-mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust.     

Severity of Xanthomonas axonopodis leaf blight on eucalypt cuttings under different levels of nutrients

Bacterial leaf blight of eucalypt, caused by Xanthomonas axonopodis, occurs mainly in forest nurseries, but also affects young plants in the field. Avoidance of leaf wetness for long periods of time over the whole nursery and using drip irrigation for clonal hedges are, currently, the most effective measures for controlling bacterial leaf blight on eucalypt. However, as these techniques are very costly, alternative management measures are required. We hypothesized that the management of mineral nutrients could reduce disease severity on eucalypts. To examine this hypothesis, rooted cuttings of Eucalyptus urophylla × E. grandis were irrigated with nutrient solutions containing different concentrations of copper (Cu), boron (Bo), potassium (K), calcium (Ca) and nitrogen (N) before inoculation with X. axonopodis 30 days after starting the nutrient solution treatments. Concentration of K higher than that recommended, commonly used in miniclonal hedges, reduced the severity of bacterial leaf blight. However, disease severity increased with an increasing of N/K ratio. The management of mineral nutrition solution, especially K, has great potential for integrated disease management of X. axonopodis in eucalypt nurseries.