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.     

Cultural characteristics and pathogenicity of Pestalotiopsis funerea on Cupressus arizonica

The fungal pathogen Pestalotiopsis funerea principally attacks conifer species, causing necrosis on infected tissues, and sometimes death of plants. The main objectives of this study were to determine optimal in vitro growth conditions for Spanish isolates of P. funerea and to evaluate the virulence of these isolates on Cupressus arizonica under field and laboratory conditions, with the goal of verifying possible correlations between these variables. Eight isolates of P. funerea derived from C. sempervirens, C. arizonica and Quercus pyrenaica were used in the assays. In the growth rate experiment, five culture media (PDA, MEA, WA, PCA and TAKAY medium) and six temperatures (5, 10, 15, 20, 25 and 30°C) were evaluated. In the pathogenicity tests, two different experiments were carried out: (i) laboratory inoculations used 30‐mm‐long twigs of C. arizonica; (ii) in field inoculations, twigs of C. arizonica were inoculated with mycelia through wounds. Four months after inoculation, length of the twigs affected by necrosis were measured. The results suggested that Spanish P. funerea isolates had optimum growth at 25°C on TAKAY medium. The fungus caused substantial necroses on C. arizonica twigs after inoculation in both excised twigs and on adult C. arizonica trees. A positive relationship between inoculations carried out under laboratory and field conditions was found. No clear relationships were found between cultural characteristics and pathogenicity for P. funerea.     

Pathogenicity of Ceratocystis resinifera to Norway spruce

The blue‐stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single‐point inoculations on young trees (two inoculations per tree on 14‐year‐old trees) and mass‐inoculations on older trees (～200 inoculations per tree on 34‐year‐old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue‐stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue‐stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non‐pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.     

Population structure of Bursaphelenchus xylophilus within single Pinus thunbergii trees inoculated with two nematode isolates

A study was performed to clarify the population structure of the pinewood nematode, Bursaphelenchus xylophilus, within single Pinus thunbergii trees after double infection of nematode populations using the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. Two nematode isolates, which had different levels or the same level of virulence, were inoculated into 6‐year‐old trees at the same or different times and then the propagated nematodes were collected from the trees after 1, 6 and 9 months. When a virulent and an avirulent isolate were inoculated into a single tree, an overwhelming propagation of the virulent isolate was observed there irrespective of the inoculation order of isolates or collection time of nematodes. However, when two virulent isolates were inoculated, propagation through the interbreeding between the two isolates was observed. In the case of the staggered inoculations with two virulent isolates, the frequency of nematodes with a PCR‐RFLP pattern of the primarily inoculated isolate increased with the time after nematode inoculations. This suggested that the population structure of B. xylophilus within a single tree varied by the virulence level of nematode populations transmitted and their transmission order.     

Editor's summary

Abstract

Characterization of the virulence of bark beetle-vectored fungi is important for assessing potential impacts of beetle outbreaks. Massive inoculation of trees with a cork borer appears to give the most accurate estimate of fungal virulence, but cork borer inoculation is time and labor intensive. In October 2003, 18 Pinus contorta var. latifolia were inoculated with a beetle-associated fungus, Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel et al., at densities of 200 and 800 cork borer holes m^?2. In July 2004 nine trees were inoculated using bark flap inoculations. The fungal-induced moisture content reduction, sapwood occlusion area and needle discoloration were similar for the 800 cork borer holes m^?2 density and for bark flap inoculations, while pathogenicity symptoms induced by the 200 cork borer holes m^?2 were less intense. Bark flap inoculations were three times faster to perform than high-density cork borer inoculations, but differences in incubation time and yearly weather variation highlight the need for further studies. The bark flap method may be an efficient alternative to using massive inoculation densities when testing the ability of specific fungi to kill hosts, while the cork borer method may be a better method to assess pathogenic symptoms or the aggressiveness of specific fungi.     

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.     

Virulence of Cryphonectria cubensis on Eucalyptus species differing in resistance

Variability in virulence were detected among geographic isolates of Cryphonectria.cubensis (Bruner) Hodges. Inoculation studies of isolates, originally cultured from Brazil, Africa and Hawaii, in provenance of Eucalyptus pellita F. Muell (resistant) and E. saligna Sm. (susceptible) showed that the variation in resistance of Eucalyptus spp. is quantitatively isolate-specific; the variation in virulence quantitatively host-specific. The practical consequences of pathogenic variability of C. cubensis and the strategies of selection and breeding of Eucalyptus for resistance is discussed.     

Virulence of two Finnish Gremmeniella abietina types (A and B)

Thirteen Finnish Gremmeniella abietina isolates of types A and B were compared for differences in their virulence. Three kinds of inoculations were made: one with conidia on young Scots pine (Pinus sylvestris) seedlings and two with mycelia on the stems and shoots of young Scots pine trees. Inoculations with conidia were carried out in August 1992 and inoculations with mycelia were carried out six times between 11 August and 20 October, 1992. The experiments were evaluated in the late spring and early summer of 1993. The results showed that there was a difference in virulence between the two types. In the conidial inoculations type A infected 34.7% and type B infected 11.0% of the inoculated seedlings. For mycelial inoculations with type A the mean canker (stems) and necrosis (shoots) lengths were 19.3 mm and 8.6 mm longer, respectively, than with type B inoculations. In shoot inoculations there also was a clear difference between the two types in the number of such inoculations where no symptoms were observed. For type B shoot inoculations there was no fungal growth in 21.5% whereas for type A inoculations the figure was only 3.7%.     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa

Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica, was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3‐monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ, did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development.     

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.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

First report of Ceratocystis huliohia causing mortality of Metrosideros polymorpha trees on the Island of Kauaʻi,Hawaiʻi USA

The fungus Ceratocystis huliohia was detected for the first time in dead and dying Metrosideros polymorpha trees on the Island of Kaua?i, Hawai?i, U.S.A. Fungal isolates were recovered from diseased trees into culture, and identities were confirmed by diagnostic qPCR assay and DNA sequencing. Growth chamber inoculations confirmed C. huliohia pathogenicity on M. polymorpha seedlings and subsequently the same fungus was recovered, completing Koch's postulates. DNA sequence analysis revealed a unique ITS haplotype for Kaua?i isolates of C. huliohia that has not been found on Hawai?i Island.     

Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.     

Tinctoporellus epimiltinus,a causal fungus of butt rot of Japanese cypress

One of the causal fungi of butt rot of Chamaecyparis obtusa in Kyushu Island, Japan, was identified as Tinctoporellus epimiltinus from cultural characters, and by mating and inoculation tests. The cultural characters of the isolates obtained from butt rot of C. obtusa and T. epimiltinus isolated from the basidiocarp tissue and from decayed wood were examined. The morphological characters and chemical reactions of the isolates from butt rot were typical for T. epimiltinus. Di‐mon mating tests were performed between five monospore isolates of T. epimiltinus and five heterokaryotic isolates from the butt rot. All of the monospore isolates examined were heterokaryotized by the isolates from butt rot. Tinctoporellus epimiltinus isolates were inoculated on roots of 24‐year‐old C. obtusa. Butt rot was observed on all inoculated trees 1–2 years after the inoculation, and the inoculated fungus was re‐isolated from the decayed parts of the inoculated trees.     

Variability of virulence of Nectria galligena towards northern hardwoods

The variability of virulence within a Québec population of the fungal pathogen Nectria galligena was studied under greenhouse conditions. Preliminary, small-scale inoculation of Betula alleghaniensis saplings confirmed that wounds have to be present for successful infection by N. galligena and showed that saplings held at 10°C exhibited a broader range in necrotic zone length than saplings held at 24 °C for 6 weeks. Results from the inoculation of B. alleghaniensis saplings with eight full-sib progeny sets of four siblings each of N. galligena showed a wide range of virulence in this pathogen. The observed variability was probably of genetic origin since, in seven cases out of eight, one of the four siblings of a given family was significantly different from at least one other sibling of the same family. Inoculation of B. alleghaniensis saplings with a population of 43 isolates originating from different host species and geographical locations further confirmed the variability of virulence within N. galligena but failed to indicate a relationship between virulence and host or geographic provenance. Inoculation of six additional deciduous tree species with a subset of nine isolates showed the polyphagous character of the pathogen, as lesions of comparable length were generally observed on five of the species tested. None of the nine isolates tested induced any necrosis on Fraxinus americana whereas Quercus macrocarpa tended to be slightly more resistant than the four remaining true species.     

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’.     

Variation in pathogenicity among the three subspecies of Phytophthora alni on detached leaves,twigs and branches of Alnus glutinosa

Pathogenicity tests were carried out on leaves, twigs and branches of Alnus glutinosa using several isolates of Phytophthora alni ssp. alni, P. alni ssp. multiformis and P. alni ssp. uniformis in vitro. Healthy fresh leaves were collected from disease‐free areas and inoculated with mycelium on agar discs or by dipping in zoospore suspensions. In addition, twigs and branches were collected from both disease‐free and disease‐affected areas, inoculated with mycelium on agar discs and incubated at four temperatures (15, 20, 25, 30°C). All subspecies tested were pathogenic but with varied level of virulence. In inoculation tests on foliage, wounding was a key factor in causing infections: lesions on inoculated wounded leaves were larger than on non‐wounded leaves. In the twig and branch inoculation tests, no differences in virulence were observed among the P. alni subspecies in terms of sampling locations, but lesions differed in size according to incubation temperature, with the largest lesions occurring on tissues incubated at 25°C. The work is the first to report foliar necrosis caused by P. alni on A. glutinosa. P. alni ssp. uniformis was the least virulent of the subspecies in branch inoculations. These findings demonstrate that various tissues of A. glutinosa could act as sources of pathogen inoculum and may disseminate alder Phytophthora in natural ecosystems.     

Fungitoxic properties of an antibiotic against dutch elm disease

A formulation containing solubilized Nystatin called Ceratocide, is fungitoxic to Ceratocystis ulmi (Buis.) Moreau as shown in laboratory studies. In controlled inoculations studies in eight-year-old elm saplings, Ceratocide was shown to have prophylactic utility in Dutch elm disease control when injected 2, 4 or 10 days before a challenge inoculation with C. ulmi. Therapeutic control was not obtained with a single injection of Ceratocide. Most infected trees succumbed to Dutch elm disease either during the same year of treatment or a year later.     

Genetic variation in dieback resistance in Fraxinus excelsior confirmed by progeny inoculation assay

Ash dieback caused by the pathogenic fungus Hymenoscyphus fraxineus [previously known as H. pseudoalbidus (sexual stage) and Chalara fraxinea (asexual stage)] is a widespread problem in Europe. Here, we assess crown damage from natural infection and necrosis development following artificial controlled inoculations on full‐sib and half‐sib progeny from Danish Fraxinus excelsior clones with contrasting and well‐characterized levels of susceptibility to the disease. The inoculation assay was performed on a total of 123 offspring, and necrosis development monitored over two years. The offspring from low susceptible mother clones developed smaller necroses when compared to offspring from susceptible clones. Their crown damage due to natural infections was also significantly less. The correlation coefficient between average crown damages of mother clones and the average of their progeny was 0.85 (natural infections), while the correlation between crown damage of mother clones and the average necrosis development in their progeny after controlled inoculation was 0.73. The correlation between resistance of parent trees and crown damage/necrosis development on their offspring confirms the presence of heritable resistance and indicates that a bioassay based on controlled inoculations has the potential of becoming a fast and cost‐effective tool for estimation of dieback susceptibility in breeding programmes for resistance in ash trees.