A rapid,apple-based test for virulence in Cryphonectria cubensis isolates

The presence of the Eucalyptus canker pathogen Cryphonectria cubensis in South Africa is of concern to the local forestry industry. Quantification of the virulence of this fungus through the inoculation of trees, is both time consuming, and expensive. In this study, the potential to use apples in screening for virulence of isolates of C. cubensis, was tested using different apple cultivars and inoculation procedures. The best indication of virulence was given on Golden Delicious apples incubated at 25°C for 14 days. Here, the lesion size associated with inoculations of C. cubensis, was found to correlate significantly with the virulence of the isolates, as determined by inoculations on trees.     

Cryphonectria canker on Tibouchina in Colombia

Cryphonectria canker, caused by Cryphonectria cubensis, has limited the development of new Eucalyptus plantations in tropical and subtropical regions. The pathogen is commonly found on Eucalyptus, but its occurrence on other hosts in the Myrtaceae has also been documented. In this study C. cubensis is reported as the causal agent of a serious canker disease on Tibouchina spp. (Melastomataceae) in Colombia. We used morphological studies, pathogenicity tests on Eucalyptus and Tibouchina, and a phylogenetic study using partial ribosomal DNA sequence data. This is the first record of C. cubensis on a host outside the Myrtaceae.     

Effect of environment on the response of Eucalyptus clones to inoculation with Cryphonectria cubensis

The ascomycete Cryphonectria cubensis causes severe losses in Eucalyptus plantations in South Africa and selection programmes for disease tolerance are necessary. The aim of this study was to use two C. cubensis inoculation trials, planted at different locations to assess the disease susceptibility of the clones and the effect of the environment on disease development. These two trials consisted each of 21 Eucalyptus clones (E. grandis, E. grandis × camaldulensis and E. grandis × urophylla). All trees were inoculated with a single virulent strain of C. cubensis and lesion widths measured 6 and 12 months after inoculation. Clones differed significantly in their tolerance to C. cubensis. Further, disease severity differed depending on the geographical location of the trial. A significant clone × locality (genotype × environment) interaction was observed. Therefore, screening for disease resistance should take place only in areas where clones will be commercially grown.     

Quambalaria eucalypti found on Eucalyptus in Indonesia

The Eucalyptus plantation industry in Indonesia has expanded rapidly during the last few decades. During routine nursery disease surveys, symptoms of a leaf and shoot blight disease were detected on Eucalyptus mother plants. Isolates were obtained from symptomatic tissues and identified using DNA sequence analyses. Phylogenetic analyses showed that the isolates were those of Quambalaria eucalypti. Pathogenicity tests were conducted with isolates of Q. eucalypti on clones of E. pellita and E. grandis × E. pellita hybrids. These resulted in symptoms similar to those observed on naturally infected plants. Eucalyptus genotypes tested showed variation in their susceptibility, highlighting the potential to select and breed for resistance and thus to manage future outbreaks of the disease. This is the first report of the pathogen in Indonesia as well as in Southeast Asia.     

Comparison of procedures to evaluate the pathogenicity of Ceratocystisfimbriata sensu lato isolates from Eucalyptus in South Africa

Ceratocystis fimbriata sensu lato (s.l.) is an important pathogen of Eucalyptus. Pathogenicity of isolates has typically been evaluated by inoculating seedlings under greenhouse conditions. It is, however, not clear how accurately this reflects pathogenicity under field conditions. In this study, five techniques to potentially screen C. fimbriata isolates for their relative pathogenicity to Eucalyptus were compared. These included: in vitro growth comparisons on artificial media; inoculations on apples; inoculation on Eucalyptus seedlings in a greenhouse; inoculations on Eucalyptus bolts freshly cut from stems of young trees; and field inoculations on young trees. Eight isolates of C. fimbriata s.l. collected from various areas in South Africa were used. There was considerable variation in growth in culture and aggressiveness of the eight isolates. Field inoculations on young trees were best correlated with inoculations of bolts (r = 0.76). Lower correlation coefficients were obtained with seedlings (r = 0.59), apple inoculations (r = 0.56), and in vitro colony growth (r = 0.42). Inoculation of bolts provides a rapid and reliable method to screen isolates of C. fimbriata s.l. for pathogenicity to Eucalyptus.     

Susceptibility of Eucalyptus grandis to Cryphonectria cubensis

Artificial inoculation of rooted Eucalyptus grandis cuttings with Cryphonectria cubensis in the glasshouse showed that significant differences exist in the susceptibility of various Eucalyptus clones to the pathogen. A 61.3% difference in susceptibility was observed between the most resistant and susceptible group of clones. Results appeared, however, to have been influenced by genotype × environment interaction.     

Pathogenic variation in Cylindrocladium quinqueseptatum causing leaf blight of Eucalyptus1

A wide range of pathogenic variation was observed among the five isolates of Cylindrocladium quinqueseptatum leaf blight (CLB) which could be distinguished as different physiologic strains on 11 differential provenances of Eucalyptus. Susceptibility ranking of different provenances to five isolates also differed significantly indicating differential interaction between isolates and provenances. Analysis of variance showed that CLB severity of a provenance is mainly governed by the genetically different isolates and also that the provenances have closer genetical relationship. The results provide the first evidence for the existence of physiologic strains in C. quinqueseptatum.     

A serious new wilt disease of Eucalyptus caused by Ceratocystis fimbriata in Central Africa

In a recent survey of Eucalyptus clones in the Republic of Congo, Central Africa, a serious wilt and die-back disease of two different hybrid clones was observed. Affected trees ranged in age from approximately 6 months to 4 years. Isolations from symptomatic plant material consistently yielded a Ceratocystis species. On the basis of morphology and sequence data this fungus was identified as Ceratocystis fimbriata, a well-known wilt and canker pathogen of many economically important plants. The Eucalyptus isolates were compared with other Ceratocystis spp. based on sequence data generated from the ITS and 5.8S region of the rRNA operon. The results confirmed the identity of the Ceratocystis isolates from Eucalyptus as C. fimbriata and showed that they group with other C. fimbriata isolates from Brazil, South Africa and Europe. Inoculations on young Eucalyptus plants were conducted in the greenhouse and all three of the Congolese isolates tested, produced typical lesions in the bark and xylem. This study represents the first report of C. fimbriata as a pathogen of Eucalyptus in Africa. This is a serious new disease that will require considerable study in order to ensure that losses, caused by C. fimbriata, do not continue.     

Identification of Phytophthora nicotianae on the aerial portion of eucalypt seedlings

The pathogenicity, physiology and biochemical features of three isolates of Phytophthora. nicotianae var. nicotianae from Eucalyptus delegatensis and E. globulus seedlings were investigated. The acid phosphatase zymograms and total protein profiles confirmed the specific identity of the 3 isolates. Despite significant differences in mycelial growth and zoosporangium production, no differences were noted in pathogenicity. Artificial inoculations revealed a variability in resistance to P. nicotianae within Eucalyptus species.     

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.     

Aggressiveness,cultural characteristics and genetic variation of Ceratocystis fimbriata on Eucalyptus spp.

Ceratocystis wilt caused by Ceratocystis fimbriata is currently one of the most important diseases affecting Eucalyptus in Brazil. This disease is controlled by planting resistant clones; however, possible variability in the pathogen population may compromise the selection of resistant genotypes. Therefore, this study aimed to evaluate the aggressiveness of C. fimbriata isolates obtained from Eucalyptus spp, as well as their cultural characteristics and genetic variation of their ITS rDNA gene region. We found a significant isolate × clone interaction, with the isolate RM35 being the most aggressive and presenting a broader spectrum of aggressiveness, causing greater xylem discoloration on a larger number of clones. This isolate is the most suitable for artificial inoculations focusing on the selection of resistant materials. Clones CLR‐236 and CLR‐212 were identified as the most resistant and clones CLR‐223 and CLR‐240 as the most susceptible and those that are recommended as reliable comparators in artificial inoculations. All isolates were morphologically similar and differed from C. fimbriata from sweet potato by the formation of a wide mouth endoconidiophore that produces doliform endoconidia. According to the culture media and temperature applied, the most favourable conditions for mycelial growth were observed using malt extract agar (MEA) and temperatures ranging from 24 to 26°C. There was no correlation between sporulation and aggressiveness. Great variation in ITS sequences was observed, and a total of five ITS genotypes were identified among the ten isolates tested.     

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.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

First report of Calonectria cerciana causing leaf blight of Eucalyptus in northern India

Eucalyptus spp. and their hybrids are frequently cloned and mass planted across farmland tracts and commercial plantations in northern India. It is a viable feeder species to the paper and pulp industries in this region. In 2018 and 2019, during field surveys conducted in northern India, a serious leaf blight disease was frequently observed in E. tereticornis plantations. Isolation from the blighted leaf samples consistently yielded fungal isolates having Calonectria‐like morphology. Morphological features coupled with sequence analysis of partial β‐tubulin (TUB2) and partial translation elongation factor‐alpha (TEF1) gene regions of two fungal isolates confirmed the species as Ca. cerciana. In detached leaf assays and glasshouse inoculation experiments, both isolates produced symptoms similar to those observed on the naturally infected leaves. Koch's postulates were fulfilled by re‐isolating Ca. cerciana from the inoculated leaves. This work is the first to confirm that Ca. cerciana is associated with a serious leaf blight disease of Eucalyptus in northern India and is an important addition to the taxonomy of Calonectria fungi in India.     

In vitro test of virulence in the progeny of a Heterobasidion interspecific cross

Annosum root rot, caused by Heterobasidion annosum s.l., is the economically most devastating disease of coniferous forests in the northern hemisphere. We have analysed virulence of the progeny isolates of a H. annosum interspecific cross between one S and one P homokaryotic isolate from North America. Virulence was measured as mortality rate among 29 pine (Pinus sylvestris) or 58 spruce (Picea abies) seedlings per homokaryotic progeny isolate. The assay showed a wide range of virulence among the 97 progeny isolates on pine and segregated as a continuous character. The heritability of virulence on pine was estimated to be 0.088 in this study. A strong correlation between virulence on pine and spruce was also found, although the range of virulence between isolates on spruce was smaller than that on pine. No correlation between high virulence and either fast radial growth rate on malt extract agar or high wood decay capacity was found.     

Virulence and double‐stranded RNA in Sphaeropsis sapinea

Forty wildtype isolates of Sphaeropsis sapinea were grouped into the morphotypes A and B based on previously defined differences in cultural and morphological criteria as well as restriction sites for Dde I and Bst UI endonucleases in nuclear ribosomal DNA amplicons. Thirteen of 20 type A isolates and nine of 20 type B isolates contained detectable dsRNA (55%) of different molecular weight and size. dsRNA was transmitted into conidia at a frequency of 71–100%. By selecting single conidia, dsRNA‐free subcultures were obtained from six of 22 isolates containing dsRNA. Pathogenicity tests on expanding buds of landscape trees of three species of Pinus showed highly significant statistical interactions between isolate virulence, Pinus species, and year. Pine species‐year had a profound impact on virulence. The pattern in the interactions was revealed by principal component analysis of the interaction sums of squares of the anova (Additive Main Effects and Multiplicative Interaction; AMMI). Pinus sylvestris was highly interactive in its susceptibility to S. sapinea with seasonal effects. P. nigra and P. resinosa were more stable. The interactivity analysis was used to apportion interaction to specific isolates to improve the accuracy of the estimates of virulence. Estimates of the relative virulence of isolates were predicted over five different Pinus species‐years. Isolates were ranked in virulence and interactivity using the AMMI model. This model permitted mean separation tests of the relative virulence among isolates over the combined Pinus species‐years. One isolate was identified as potentially having dsRNA‐mediated hypovirulence based on the significantly greater virulence of its isogenic, dsRNA‐free subculture, as expressed over the three Pinus species and 2 years. Type A isolates containing dsRNA ranged from stable to highly interactive and from low to high in virulence. Type B isolates containing dsRNA were similar in interactivity but virulence ranged from avirulent to moderate, seldom exceeding the mean for S. sapinea. dsRNA‐free isogenic subcultures tended not to express higher virulence than their dsRNA‐containing parent strains but often changed in interactivity. Therefore, in one year a dsRNA‐free subculture might be more virulent than its dsRNA‐containing parent. In another year the dsRNA‐free subculture might be less virulent.     

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.     

Mycosphaerella species associated with leaf disease of Eucalyptus globulus in Ethiopia

Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region.     

Phytophthora ramorum is a generalist plant pathogen with differences in virulence between isolates from infectious and dead‐end hosts

Variation in virulence was examined among isolates of Phytophthora ramorum from epidemiologically important or infectious (non‐oak) and transmissive dead‐end (oak) hosts from North America. Twelve isolates representative of the genetic, geographic and host range of P. ramorum in the western United States were inoculated on leaves of Umbellularia californica (bay laurel or bay) and stems of Quercus agrifolia (coast live oak). In spite of extreme genetic similarity among the isolates employed, and even within the same genotype, significant differences in lesion size were measured, suggesting virulence in this pathogen is also controlled by epigenetic factors. A strong positive correlation between lesion size on bay laurel and coast live oak provides experimental evidence P. ramorum is a generalist pathogen that lacks host specificity. Isolates from non‐transmissive oaks were significantly less pathogenic both on oaks and bays than isolates from infectious hosts. These results are essential to further our understanding of the epidemiology and evolutionary potential of this pathogen. A quantitative differential in virulence of isolates from hosts with different epidemiological roles has been described for many animal diseases, but is a novel report for a plant disease.     

黑木相思与速生桉商品用材林培育的经济效益对比分析

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