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.     

Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

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.     

Cultural characteristics and pathogenicity of Melanconium juglandinum

Cultural characteristics and pathogenicity of Melanconium juglandinum, the agent of the black pustular dieback of Juglans spp. in Europe were investigated. Melanconium juglandinum was consistently isolated from diseased twigs and branches of Persian (English) walnut trees (Juglans regia) in Central Italian Apennine valleys. In growth and conidia germination tests, the fungus was shown to be active within a broad range of temperatures (5–30°C), with the optimum being between 20 and 30°C. Monthly artificial inoculations performed over a period of 2 years with mycelial plugs on 2-year-old seedlings of Persian (English) walnut developed symptoms within 15 days with maximum canker length occurring when inoculation was carried out between June and August. Koch's postulates were fulfilled and M. juglandinum was proved to be a virulent pathogen.     

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.     

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.     

Pathogenic variability of various isolates of Seiridium cardinale,S. cupressi and S. unicorne inoculated on selected Cupressus clones and seedlings

Comparative studies of the pathogenicity of eight isolates of three Seiridium species were carried out by stem inoculations on Cupressus sempervirens and C. glabra seedlings and clones. Seiridium cardinale and S. cupressi caused bigger and more severe cankers than all strains of S. unicorne. During the first nine months after inoculation, the canker length of S. cardinale was larger than the cankers of S. cupressi. However, during the second year after inoculation, S. cupressi cankers were larger and more severe than those of 5. cardinale.     

Studies of Marssonina castagnei in Australasia

Comparative studies of conidium morphology and host range and pathogenicity tests of Australian and New Zealand isolates of Marssonina castagnei in the laboratory established their equivalence which was indicative of a common origin. It is speculated that trans-Tasman dispersal is responsible for the introduction of M. castagnei into New Zealand and its widespread distribution. This is based on the prior recording of M. castagnei in Australia, the potential of M. castagnei conidia for long distance transport and the proven records of trans-Tasman dispersal of other pathogens. Laboratory inoculations and field observations showed P. alba cv. Silver poplar and its hybrids to be highly susceptible to M. castagnei. Populus alba X P. glandulosa hybrids and Italian selections of P. alba showed high field resistance.     

Pathogenicity of Spanish isolates of Heterobasidion annosum s. s. in Pinus pinaster seedlings

The aim of this study was to test the pathogenicity of two Spanish isolates of Heterobasidion annosum sensu stricto in 2‐year‐old Pinus pinaster seedlings. Two types of inocula (woodchips and sawdust) were used to infect the seedlings by two different routes (stem inoculation and soil infestation). The mortality rates of the stem‐inoculated seedlings differed significantly from controls, but those of the seedlings infected via soil infestation did not differ. For both types of inoculation, the lesions were longer, and wilting symptoms were more severe in the seedlings inoculated with H. annosum than in control seedlings. For stem inoculation, biomass allocation did not differ significantly between the infected and control seedlings. However, the percentage of fine roots was lower in seedlings infected via soil infestation than in the control seedlings. To our knowledge, this is the first pathogenicity test with H. annosum isolates and P. pinaster.     

Growth response and nutrient utilization of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> seedlings inoculated with bioinoculants under tropical nursery conditions

We investigated the role of tetrapartite associations between an arbuscular mycorrhizal (AM) fungus (Glomus geosporum), phosphate solubilizing bacteria (Paenibacillus polymyxa), Frankia and Casuarina equisetifolia on growth, nutrient acquisition, nutrient utilization and seedling quality of C. equisetifolia. Seedlings of C. equisetifolia were grown in an Alfisol soil and inoculated with G. geosporum, P. polymyxa and Frankia either individually or in combinations. Inoculation of bioinoculants stimulated seedling growth, the efficiency of nutrient uptake and improved seedling quality. However, microbial inoculation generally reduced the efficiency of nutrient utilization in dry matter production (nutrient use efficiency). Inoculation of P. polymyxa or Frankia increased the extent of AM colonization, which resulted in the accumulation of the nutrients. Seedlings inoculated with Frankia and G. geosporum had more, and heavier nodules compared to seedlings inoculated with Frankia alone. Dual inoculation of microbes was more effective than individual inoculations. The growth response of seedlings to inoculation involving all the microbes was greater than the response to either individual or dual inoculations. The results of this study showed that the tetrapartite association could improve the growth, nutrient acquisition and seedling quality of C. equisetifolia under tropical nursery conditions.     

Pathogenicity to alder of Phytophthora species from riparian ecosystems in western Oregon

Described as one of the most destructive pathogens of agricultural crops and forest trees, Phytophthora is a genus of microorganisms containing over 100 known species. Phytophthora alni has caused collar and root disease in alders throughout Europe, and a subspecies has recently been isolated in North America. Reports of canopy dieback in red alder, Alnus rubra, prompted a survey of their overall health and to determine whether P. alni was present in western Oregon riparian ecosystems. Over 1100 Phytophthora isolates were recovered, representing 20 species and 2 taxa. Phytophthora‐type cankers were observed in many trees, and their incidence was positively correlated with canopy dieback. High levels of mortality for red alder were not observed, which suggests these Phytophthora species are not aggressive pathogens. To test this hypothesis, three stem wound inoculations and one root dip were conducted on red alder seedlings using 13 Phytophthora species recovered from the riparian survey. Ten of the 13 Phytophthora species produced significant lesions in at least one pathogenicity test. Phytophthora siskiyouensis produced the largest lesions on red alder from the two stem wound inoculation tests conducted under summer conditions, while P. taxon Pgchlamydo caused the largest lesions during the winter stem wound inoculation test. Phytophthora gonapodyides, P. taxon Pgchlamydo and P. siskiyouensis have previously been found associated with necrotic alder roots and bole cankers in the field, and with the pathogenicity results reported here, we have established these species as causes of Phytophthora root disease and Phytophthora bole canker of alder in Oregon. While none of the Phytophthora species were especially aggressive towards red alder in the pathogenicity tests, they did cause localized disease symptoms. By weakening the root systems or boles of alders, the Phytophthoras could be leaving alders more susceptible to other insects and pathogens.     

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.     

Relative susceptibility of oaks to seven species of Phytophthora isolated from oak forest soils

Isolates of Phytophthora cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercetorum and two unidentified species were tested for their pathogenicity to eastern US oak species by root and stem inoculations. Experiments were conducted during two different periods and included 1‐, 2‐ and 20‐year‐old oaks grown under greenhouse and field conditions. Species of Phytophthora were pathogenic in varying degrees to the oak species tested. All species were pathogenic to fine and taproots of at least one oak species. The fine root damage caused by the species of Phytophthora ranged from 9 to 55% when compared to the controls. Roots were more susceptible during the fall inoculation period than the summer. With exception of Phytophthora sp1 and P. quercina‐like, all species of Phytophthora were pathogenic to oak stems with P. cinnamomi and P. citricola being the most aggressive. Quercus montana and Q. rubra were the most susceptible oak species to stem inoculation. Lesion sizes were considerably larger when 20‐year‐old trees were inoculated. Generally, no significant differences in lesion sizes were detected in greenhouse tests when the summer and fall inoculation periods were compared. However, on 2‐year‐old field‐grown seedlings, lesion sizes were considerably smaller or not significantly different from controls during the fall inoculation period, suggesting lower, late season temperatures may restrict lesion development.     

Discula quercina as a possible causal agent of dieback on oak trees in Iran

During spring and summer of 2016, dieback symptoms including blights of leaves, twigs, and acorns were observed on current year shoots of Quercus infectoria in the Zagros oak forests of Iran. The fungus isolated from damaged tissues was identified as Discula quercina based on both morphological characteristics and ITS sequencing. To fulfil the Koch's postulates, a representative isolate was inoculated onto shoots of Q. infectoria and Quercus libani in both laboratory and forest conditions. Responses of the two oak species to inoculation with D. quercina were examined under laboratory and forest conditions in a completely randomized experiment. Discula quercina was clearly involved in oak dieback, and Q. infectoria was more susceptible than Q. libani to damage by the pathogen. This is the first record of the occurrence and pathogenicity of the fungus Discula quercina on Quercus infectoria. The fungus is considered as an emerging pathogen on oak trees in Zagros forests in Iran. Furthermore, the pathogenicity of the Discula quercina on Q. libani under laboratory and forest conditions increases the potential importance of this pathogen in Zagros forests.     

Pathogenic ice‐nucleation active bacteria in willows for short rotation forestry

Extensive sampling of endophytically growing bacteria from shoots of diseased Salix viminalis clones were performed in trials at two experimental sites in central Sweden. Field, greenhouse and laboratory studies were conducted to identify and characterize the plant‐associated bacteria and determine their possible role in the severe dieback appearing in short rotation forestry (SRF) plantations in Sweden during the last few years. The most frequently isolated types were found to be non‐fluorescent Pseudomonas fluorescens (biotype A, C, G) and Sphingomonas spp. (23%). Erwinia spp., P. fluorescens, P. syringae and Xanthomonas spp. each constituted 11–14% of the bacterial isolates respectively. After biochemical characterization and screening for ice‐nucleation activity (INA) 78 of the originally 279 isolated bacterial strains were selected for pathogenicity tests and further analyses. Of the 78 selected isolates, 95% were found to be pathogenic, giving different degrees of necrotic reactions after inoculation to Salix plants. Half of these pathogens (46%) also showed strong INA (at temperatures between ?2.5 and ?5°C). With respect to both isolation frequency, clonal distribution in the field, ice‐nucleation ability and necrotic reactions of willow in laboratory tests, we consider the following types to be the most important pathogens in the field: Erwinia spp., P. syringae, Sphingomonas/P. fluorescens spp. (biotype A, C, G) and Xanthomonas spp. Reported damage and dieback in many SRF‐plantations are, according to our results, very likely to be a consequence of bacterial infections, particularly of pathogenic INA‐strains, which both initiate freezing stress and aggravate the frost damage of plants by subsequent invasion of plant tissues leading to complete death of plants under field conditions.     

Characterization and pathogenicity of six Cytospora strains causing stem canker of wild apple in the Tianshan Forest,China

Cytospora species are capable of causing destructive cankers of stems belonging to a wide range of woody plant species. In severe cases, cankers may lead to dieback of twigs and branches. Little is known about the Cytospora species causing canker disease of wild apple (Malus sieversii) trees in the Wild Fruit Forest Reserve in Tianshan Forest, Xinjiang Uygur Autonomous Region, China. In this study, six Cytospora isolates belonging to two species were isolated from cankerous lesions of wild apple twigs. Based on multi‐locus phylogenetic analysis using three DNA markers (ITS, tef1‐α and tub2) and morphological characterization, these isolates were identified as Cytospora mali and Cytospora parasitica. Temperature trials (15, 20, 25 and 30°C) showed that the optimal growth temperature for six isolates was 25°C. At a variety of temperatures, C. mali isolates tended to grow faster than isolates of C. parasitica, with the C. mali isolate, EGI1 performing better than others with regard to growth rate. Morphological observations showed that these species exhibited a single locule without conceptacles, and the conidia length was 3–5 μm. In vitro inoculation trials of twigs and leaves of M. sieversii seedlings revealed that the C. mali isolates were highly virulent phytopathogenic fungi, whereas the C. parasitica isolates were less virulent. The isolate EGI1 was the most virulent isolate among the six isolates. This paper presents the first report of pathogenic Cytospora spp. of the M. sieversii Tianshan Wild Fruit Forest Reserve of Yili, Xinjiang in China. It will aid in the understanding of how apple tree cankers are induced and provide disease management guidelines for M. sieversii forest conservation.     

Biscogniauxia nummularia: pathogenic agent of a beech decline

Decline of European beech (Fagus sylvatica) in Sicily and Calabria (Italy) was observed to be associated with the ascomycete Biscogniauxia nummularia. This fungus is naturally present in beech stands and to date has not been considered a primary pathogenic agent. Artificial inoculations were performed to assess its pathogenicity on beech and to determine if Sicilian and Calabrian isolates differ in virulence. Biscogniauxia nummularia played an evident primary pathogenic role under the environmental conditions studied. Virulence of the isolates was variable and did not depend neither on the geographic origin nor the environmental conditions at the sites where pathogenicity testing was performed.     

Pathogenicity of seed‐associated fungi to Podocarpus falcatus in vitro

Twenty‐nine fungi that were isolated from seeds and female cones of Podocarpus falcatus from natural forests in Ethiopia, were assessed for their impact on seeds and seedlings of the same host. Based on the results from in vitro seed inoculation tests, we could group the fungi into five categories as: (i) isolates that were pathogenic only to seeds and had no obvious impacts on the germlings; (ii) isolates that were pathogenic only to the germlings; (iii) isolates that were pathogenic both to seeds and the emerging germlings; (iv) isolates that were more or less harmless; and (v) isolates that were germination promoters. Inoculation tests were also performed on 4–7‐day‐old aseptically grown seedlings. Fusarium oxysporum and Polyporus sp., were strongly pathogenic to both seeds and seedlings, while Nectria gliocladioides, Peniophora cinerea and Pestalotiopsis neglecta also demonstrated pathogenicity but to a lesser extent. Other isolates, e.g. Diaporthe spp. resulted in increased germination of P. falcatus seeds and no pathogenicity to seedlings. However, further investigations are required in order to find out how these fungi behave under nursery or field conditions.     

Physiological and morphological variation of Gremmeniella abietina from Spain

Gremmeniella abietina isolated from Pinus halepensis in Spain was characterized based on disease symptoms and conidia morphology. The disease symptoms, which became more evident in the spring, included drying up of needles and branches with some distortion of terminal twigs, resulting in dieback and sometimes to death of trees of all sizes. Variation in conidia size and growth rate was investigated between 15 isolates from Spain, and the conidia morphology was compared with four isolates from Finland, four from Switzerland and three from the US. Great variation occurred between Spanish isolates both in growth rates and in conidia size. The growth rates of the isolates were greatest on malt agar amended with pine needle extract and at 15°C. The length and the width of the conidia of the isolates from Spain ranged between 10.7–44.8 and 1.5–4.4 μm, and most of them had three septa. The results suggest that the isolates from Spain do not belong to the Alpine biotype, and the disease symptoms caused by these isolates resembled those of the European biotype.     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.