Influence of environmental,host and cultural factors on conidium morphology of Marssonina species pathogenic to poplars

Laboratory studies investigating the influence of environmental, host and cultural factors on conidium morphology of isolates of Marssonina brunnea, M. castagnei and M. populi established that conidial features (length and breadth dimensions, shape, conidium ratios, L:B, LS:B and septum location) were remarkably stable. Significant differences in length and breadth dimensions and ratios were induced by culture media and host but not by media pH, incubation temperature, photoperiod, leaf age, leaf surface and infection severity. Significant differences in conidial dimensions and ratios of M. brunnea were observed following examination of monthly collections from a single field-grown tree. The variation in conidial features induced by environmental, host and cultural factors in no instance obscured taxonomic differences between species.     

松球壳孢菌菌落及孢子形态学对比研究

通过79个辽宁松球壳孢菌菌株与4个A型和B型标准菌株、5个美国菌株在菌落和分生孢子形态方面的对比研究，发现辽宁的菌株在形态上基本一致。辽宁菌株与美国的类型A相近，主要表现在孢子表面平滑，通常无隔或有1个隔，在成品PDA培养基上产生绒毛状、白色至灰色菌落。不同之处在于，辽宁菌株在自制PDA培养基上形成贴在培养基表面的菌落——与类型B相似，分生孢子长度介于A型和B型之间或与后者相近。5个美国菌株与类型A一致。同时发现，培养基的来源对菌落形态研究非常重要；光照使菌落气生菌丝有所减少且出现绿色；培养基和寄主种类对孢子大小有影响。     

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%.     

Variation among New Zealand isolates of Sphaeropsis sapinea

Sphaeropsis sapinea is a pathogen of many coniferous species and causes significant losses to the plantation forestry industries of many countries. New Zealand isolates of S. sapinea were examined for colony and morphological characteristics, and pathogenicity to Pinus radiata seedlings. Considerable variation was observed in colony growth form and colour, and in the growth rates on four agar media. The conidial dimensions, carbon and nitrogen utilization, and pathogenicity also varied among the isolates. The variation observed was such that the isolates could not be placed within previously described distinct morphotypes and the present results concur with previous studies cautioning the use of morphological criteria alone to partition the species. These results contrast markedly with previous reports and seemingly indicate a change in the New Zealand S. sapinea population. This difference may reflect the re‐introduction or spread of additional isolates or a shift in the structure of the resident population.     

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.     

Agrobacterium pusense,a new plant tumour‐inducing pathogen isolated from Lawson cypress

Lawson cypress (Chamaecyparis lawsoniana), an important landscape tree, is widely planted in gardens and parks throughout Iran. Crown gall disease on Lawson cypress trees was observed in Sari and Juybar Counties, Mazandaran province, northern Iran, in 2017. Isolation from galls on potato dextrose agar (PDA) containing CaCO₃ yielded bacterial colonies, the predominant types of which were purified and selected for characterization. The isolates were Gram‐negative, oxidase positive, able to grow in 2% NaCl and produced 3‐ketolactose. They hydrolysed esculin, casein and arbutin but not starch, gelatin or Tween 80. Two representative isolates were selected for PCR amplification and sequencing of DNA gyrase subunit B (gyrB) gene. In the phylogenetic tree based on the partial sequence of the gyrB gene, isolates KH1 and KH2 clustered with Agrobacterium pusense. The pathogenicity of all isolates was confirmed by inoculation on Jimsonweed (Datura stramonium) and carrot discs (Daucus carota). Confirmation of the presence of genes involved in pathogenicity was made by performing PCR with the virD2A/virD2C and VCF/VCR primer pairs which resulted in amplification of the expected 224 and 730 bp fragments in all studied isolates, respectively. A. pusense was therefore identified as the causal agent of crown and stem gall of Lawson cypress. This appears to be the first report on the natural occurrence of crown gall disease on Lawson cypress and the first record of a plant disease caused by A. pusense.     

Gremmeniella abietina types cannot be distinguished using ascospore morphology

Twenty‐one Gremmeniella abietina var. abietina type A and 12 type B samples with ripened apothecia were collected in Sweden and Finland. Morphological dimensions of the ascospores were determined with an image analyser (Leica Qwin). Measured variables included length, width, perimeter and roundness of ascospores. Variations in these variables overlapped between the two types. Type A ascospores were significantly shorter (mean length 15.8 μm) than the type B ascospores (mean length 17.1 μm). Type A ascospores were also significantly rounder than type B ascospores. Differences between these morphological characters were, however, too small to distinguish the types as separate taxonomical species. Round‐pointed and acute ascospores were found in the same apothecium but in different asci. According to the hypervariable marker GAAA1000, both round‐pointed and acute ascospores originated from the same two parents in normal segregation.     

Mycorrhizal and associated fungi of Sitka spruce in Irish forest mixed stands

Identity of mycorrhizas and isolation of symbionts and associated fungi from Sitka spruce growing in pure and mixed stands with either Japanese larch or Lodgepole pine are described and compared. More mycorrhizal types and sporocarps of the Agaricales were collected from mixed stands. Mycorrhizas of Lactarius rufus, Paxillus involutus and Suillus spp. were more prevalent on roots from mixed stands. The most common unidentified mycorrhizal type (type B) had features similar to synthesized mycorrhizas of two Basidiomycete isolates. Suillus grevillei and three un-identified types were associated specifically with Japanese larch. The main associating fungi were Oidiodendron sp. and Mycelium radicis atrovirens. The association of a “nurse” tree with Sitka spruce provides a more diverse mycorrhizal flora, the majority of which are shared between the tree species.     

Morphological and cultural variation of Seiridium spp. from cankered Cupressaceae hosts in New Zealand

Studies of Seiridium. spp. isolated from cankered cypresses and related hosts in New Zealand indicate that only 2 species are present, namely, 5. unicorne and 5. cardinale, following sdtton, and not 3 species as proposed by boesewinkel. Apart from a difference in the presence/absence of conidial appendages, the two pathogens are similar in many biological characteristics. Isolates of S. unicorne showed a continuum of variation in conidial size and cultural morphology making it difficult to justify splitting of the species.     

Molecular and biological characterization of new isolates of <Emphasis Type="Italic">Cydia pomonella</Emphasis> granulovirus from Iran

The Cydia pomonella granulovirus (CpGV) is a very effective biological control agent of the codling moth, Cydia pomonella L. (Lep.: Tortricidae). Only a few CpGV isolates originating from Mexico (M), England (E), and Russia (R) have been described so far. In a field survey at different locations in Iran, CpGV isolates were collected from single or pooled codling moth larvae. The isolates, designated I1, I7, I8, I15, I22, I28, I30, I66, I67, I68, and I70 showed genetic (DNA restriction endonuclease profiles) and biological (bioassays) differences. Most isolates could be attributed to genome types similar to those found in CpGV-M, -E, and -R. Some of them were clear mixtures of different genotypes. Thus, the CpGV isolates found in the North–West of Iran make an important contribution to the known diversity of CpGV. The occurrence of novel, naturally occurring CpGV isolates emphasize the necessity of further studies towards the diversity and evolution of CpGV.     

PCR‐RFLP markers identify three lineages of the North American and European populations of Phytophthora ramorum

Phytophthora ramorum, the cause of sudden oak death and ramorum blight, has three major clonal lineages and two mating types. Molecular tests currently available for detecting P. ramorum do not distinguish between clonal lineages and mating type is determined by cultural methods on a limited number of samples. In some molecular diagnostic tests, cross‐reaction with other closely related species such as P. hibernalis, P. foliorum or P. lateralis can occur. Regions in the mitochondrial gene Cox1 are different among P. ramorum lineages and mitochondrial genotyping of the North American and European populations seems to be sufficient to differentiate between mating types, because the EU1 lineage is mostly A1 and both NA1 and NA2 lineages are A2. In our study, we were able to identify P. ramorum isolates according to lineage using polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) of the Cox1 gene, first by using ApoI to separate P. ramorum from other species and EU1 from North American populations, and then AvaI to distinguish between NA1 and NA2 genotypes. However, P. foliorum had the same restriction profile as P. ramorum NA1 isolates.     

Dothistromin biosynthesis genes allow inter‐ and intraspecific differentiation between Dothistroma pine needle blight fungi

Dothistroma septosporum and D. pini are the causal agents of Dothistroma needle blight (DNB) of Pinus spp. in natural forests and plantations. The main aim of this study was to develop molecular diagnostic procedures to distinguish between isolates within D. septosporum, for use in biosecurity and forest health surveillance programmes. This is of particular interest for New Zealand where the population is clonal and introduction of a new isolate of the opposite mating type could have serious consequences. Areas of diversity in the dothistromin toxin gene clusters were identified in D. septosporum (51 isolates) and D. pini (6 isolates) and used as the basis of two types of diagnostic tests. PCR‐restriction fragment length polymorphism (RFLP) of part of the dothistromin polyketide synthase gene (pksA) enabled distinction between two groups of D. septosporum isolates (A and B) as well as distinguishing D. septosporum and D. pini. The intergenic region between the epoA and avfA genes allowed further resolution between some of the A group isolates in RFLP assays. These regions were analysed further to develop a rapid real‐time PCR method for diagnosis by high‐resolution melting (HRM) curve analysis. The pksA gene enabled rapid discrimination between D. septosporum and D. pini, whilst the epoA–avfA region distinguished the New Zealand isolate from most other isolates in the collection, including some isolates from DNB epidemics in Canada and Europe. Although this study is focused on differences between the New Zealand isolate and other global isolates, this type of diagnostic system could be used more generally for high‐throughput screening of D. septosporum isolates.     

我国四个自然保护区森林土壤中苏云金芽孢杆菌的分布*

从我国四个不同立地带－长白山，百花山，鼎湖山，尖峰岭自然保护区，采集到林下０－５ｃｍ土层土壤样品７５个，分离到苏云金芽孢杆菌３９株。其中芽孢杆菌的总数量和苏云金芽杆菌的出土率和分离率所有差异，ｐＨ偏中性和含水量稍高的土壤，菌数较高；而与所测的各种土壤养分无明显相关。对３９株苏云金芽孢杆菌的鉴定：１７株属库尔斯塔亚种，６株属松Ｚｈｕ亚种，１６株未定名。毒力测定表明：对杨扇舟蛾和马尾松毛虫幼虫的致死率     

Low genetic diversity but frequent sexual reproduction of the chestnut blight fungus Cryphonectria parasitica in Azerbaijan

Chestnut blight was first recorded in Azerbaijan on the native European chestnut (Castanea sativa) in 2004, and since then, the disease is expanding in the country. In this study, chestnut blight was detected in seven of eight forest districts that were inspected. To characterize the local population structure of the chestnut blight fungus, 199 Cryphonectria parasitica isolates were collected and assessed for vc type, mating type and microsatellite genotype. Among these isolates, one dominant vc type was detected comprising 93% of the isolates. Six additional vc types were identified among the other isolates. The microsatellite analysis revealed a very similar pattern with 96.5% of the isolates belonging to the same multilocus genotype. Both mating types of C. parasitica were present in all seven districts with a mating type ratio not different from 1:1 in five districts. In accordance with the occurrence of both mating types, sexual fruiting bodies (perithecia) of C. parasitica were found in all districts with an overall prevalence on 20.6% of the cankers. The dominant vc type and microsatellite genotype in Azerbaijan do not occur in Europe, but have previously been found to be widespread in neighbouring Georgia. Our study reveals that sexual reproduction in the invasive C. parasitica population in Azerbaijan is frequent, although the population shows a low genetic diversity. This could favour the biological control of chestnut blight using hypovirulence, which so far does not seem to be present in Azerbaijan.     

Identification of Kenyan Armillaria isolates by cultural morphology,intersterility tests and analysis of isozyme profiles

Three groups of morphologically distinct isolates were observed among nine Kenyan Armillaria isolates based on their mycelium and rhizomorphs characteristics. Seven of the isolates were interfertile with testers of North American biological species III, VII and IX. However, tests with benomyl segregants BEN 433, BEN 157-10 and BEN AVK were intersterile with the same testers and also with the European A. mellea, A. lutea and A. ostoyae. The analysis of isozyme profiles showed that morphologically similar isolates had similar isozyme profiles of esterases. Their profiles however differed from those of the European A. mellea, A. lutea and A. ostoyae. On the basis of intersterility tests and analysis of isozyme profiles, the Kenyan isolates should be considered different.     

Diversity of Cryphonectria parasitica in western Spain and identification of hypovirus‐infected isolates

Isolates of the chestnut blight fungus Cryphonectria parasitica were obtained from 44 localities in four provinces in Western Spain and characterized for vegetative compatibility (vc) types, mating types and the presence of Cryphonectria hypovirus 1 (CHV1). Among the 1232 isolates recovered from chestnut blight cankers, 11 vc types were identified: five known vc types included in EU1 to EU74 (EU1, EU11, EU12, EU28 and EU66) and six unknown vc types (CL4, CL5 CL6, CL8, CL9 and CL10). The number of vc types found per province varied between two and seven. The vc type EU11 was present in all provinces and accounted for 48.9% of all isolates. EU1 was detected in three provinces and accounted for 39.1% of the isolates. The vc types EU12, EU66, CL5 and CL6 were present in one or two provinces and comprised between 2.4% and 3% of the isolates. The other vc types were represented by only one or very few isolates. The mating type MAT‐1 was largely dominant in the provinces Leon and Avila, while both mating types MAT‐1 and MAT‐2 were found in Salamanca and Zamora. Fourteen hypovirus‐infected C. parasitica isolates were found, nine were in vc type EU1 and five in EU11, and they were detected only in the province León. All isolates analysed contained the French hypovirus subtype CHV1‐F1.     

Determination of vc and mating types of Cryphonectria parasitica isolates by multiplex PCR and their genetic diversity in 13 chestnut-growing provinces of Turkey

Vegetative compatibility (vc) and mating types and genetic diversity of Cryphonectria parasitica isolates were determined using 183 isolates obtained from 215 infected chestnut trees growing in 13 provinces of Turkey. Based on the cultural aspects, 143 of these isolates were evaluated as virulent whereas the remaining 40 isolates were hypovirulent. When vc types of 183 isolates were classically differentiated, 135 of them matched to EU-1 (82.3%), 29 of them to EU-12 (17.6%) vc type, whereas 19 of them did not match to the two. When molecular vic markers were used, all the isolates were assigned to two EU vc types; 149 to EU-1 (81.4%) and 34 (18.5%) to EU-12. Of the majority of the isolates, 134 (73.2%) had mating-type MAT-1, while 44 (24%) isolates had MAT-2 and 5 (2.8%) isolates had both mating types. The population analysis based on two DNA marker systems, Inter-Primer Binding Site and Start Codon Targeted Polymorphism, showed no intraspecific genetic variation among the C. parasitica isolates. The prevalence of two dominant vc types revealed by this study shows that biological control with hypovirulent EU-1 and EU-12 isolates will be significant for the country. The results might be helpful to chestnut breeders carrying out resistance breeding studies to manage this disease based on hypovirulence attributed to Cryphonectria hypovirus 1.     

Genetic characterization of Botrytis cinerea isolates collected from pine and eucalyptus nurseries in Bio‐Bio Region,Chile

A total of 55 Botrytis cinerea isolates collected from Pinus radiata and Eucalyptus globulus plants cultivated in nurseries located in the Bio‐Bio Region, Chile, as well as isolates collected from native plants such as Rubus spp and Aristotelia chilensis located near the nurseries were genetically characterized. All isolates carried the Bc‐hch2 allele, thus belonging to genetic Group II, which is now referred to as B. cinerea. Genotyping based on the presence of transposons Boty and Flipper showed differences between isolates related to the plant host. Thus, transposa isolates (containing both transposons) were detected in P. radiata and E. globulus, while vacuma isolates (containing neither transposon) were detected in all plants except E. globulus. Notably, boty isolates (containing just the Boty transposon) were detected at high frequencies in all plant hosts. Analyses to detect mutations involved in resistance to fungicides such as benzimidazoles (BZ), dicarboximides and QoIs also showed differences in the studied isolates. Isolates collected from E. globulus were shown to carry mutations for all tree fungicides, while those collected from P. radiata presented mutations involved in resistance to BZ only. Isolates collected from native plant hosts did not carry any of the mutations analysed.     

Distribution of chestnut blight and diversity of Cryphonectria parasitica in chestnut forests in Bulgaria

We surveyed chestnut stands at 18 sites in 11 locations in Bulgaria in 2005 and 2007 for the presence of chestnut blight. We found chestnut blight in seven locations (Belogradchik, Berkovitsa, Brezhani, Barziya, Govezhda, Petrich and Petrovo) but not in four others (Tsaparevo, Kresna, Dupnica and Botevgrad). We successfully isolated Cryphonectria parasitica from cankers on 606 trees with symptoms of chestnut blight and assayed them for vegetative compatibility (vc) types and mating type. Three vc types were identified among the 606 isolates; all three were among the European vc types with known vegetative incompatibility (vic) genotypes. Vc type EU‐12 was the most common, representing 80% of the isolates, and was found at all locations with blight, with the exception of Belogradchik in north‐west Bulgaria, where all isolates were vc type EU‐2. Only one population (Barziya) had more than one vc type, with a combination of EU‐12 and EU‐10 in almost equal frequencies. Similarly, the diversity of mating types was very low. All but three of 536 isolates assayed were in mating‐type MAT‐1; MAT‐2 was only found in one population in the north‐west (Berkovitsa). We inspected 671 bark samples from chestnut blight cankers with stromata of C. parasitica and found perithecia in only 33, of which 28 were from Berkovitsa where MAT‐2 was present. We did not detect hypoviruses in any of the 270 isolates screened using the standard double‐stranded RNA extraction protocol. Similar to results from previous studies in south‐eastern Europe, the diversity of vc types and mating type of C. parasitica in Bulgaria is low, and reproduction of the fungus is mainly asexual. Unfortunately, naturally occurring hypovirulence was not detected. Nevertheless, we observed a small number of superficial cankers typical of those caused by C. parasitica isolates infected with a hypovirus.