Association of Sphaeropsis sapinea with insect‐damaged red pine shoots and cones

The association of the shoot blight and canker pathogen Sphaeropsis sapinea with red pine (Pinus resinosa) shoots and cones damaged by insects (especially Dioryctria sp.) was investigated. Samples from a single plantation approximately 35 years old, in Sauk Co., Wisconsin and also from three plantations, between approximately 40 and 50 years old, located in an area of pine shoot moth activity in the preceding year in Adams Co., Wisconsin were visually examined. Samples were arbitrarily collected from trees felled in the first plantation in May. Pycnidia of S. sapinea and insect damage were observed on 56 of 91 (62%) of closed cones and 17 of 165 (7%) of previous year's shoots. In the absence of insect damage, pycnidia of the pathogen were identified only on eight of 91 (9%) closed cones and never on previous year's shoots. In each of the other three plantations, 10 trees were located at intervals along transects in mid‐June; one branch from the lower half of the crown per tree was pruned off, and both current and previous year's shoots were examined. Insect damage and S. sapinea pycnidia were too rare on current year's shoots to draw any conclusions. Insect damage occurred on 20–40% of over 2000 previous year's shoots that were examined, but pycnidia of the pathogen were identified on only about 5%. Although infrequent, S. sapinea was identified in association with insect‐damaged previous year's shoots from these three plantations three times more frequently than those without insect damage. Random amplified polymorphic DNA (RAPD) markers from eight randomly selected isolates were consistent with the A group of S. sapinea, which can be aggressive on red pine. This ability to exploit insect‐damaged shoots may facilitate long‐term persistence of S. sapinea at low disease incidence and severity. The potential role of insect wounds as infection courts and insects as vectors of this important pathogen of pines deserves further study.     

Site-related influences on cone-borne inoculum and asymptomatic persistence of Diplodia shoot blight fungi on or in mature red pines

The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata sporulate abundantly on cones of many pine hosts. Variation in incidence and abundance of potential inoculum from cones and frequency of asymptomatic persistence on or in shoots was examined for mature red pines in sites differing in dominant presettlement vegetation and soil type in Bayfield and Douglas counties in northern Wisconsin. Collections were made in each county from 6 plantations, 3 each in areas historically vegetated with jack pine and soils mapped as sands and three in areas historically vegetated with red pine with soils mapped as loamy sands. At each site, 5 cones were collected from each of 5 red pines and 10 shoots were collected from up to 5 red pines. Conidia from cones were quantified with a water wash and filtration technique. Diplodia species were cultured from surface-disinfested asymptomatic shoots. A species-specific PCR assay was used to identify the Diplodia species from cones and shoots. Although cones and asymptomatic shoots from each county yielded D. pinea and D. scrobiculata, D. pinea was detected more frequently. More conidia were obtained from cones from Douglas Co., where there is a history of severe shoot blight damage, than cones from Bayfield Co. In Douglas Co., more conidia were obtained from cones from plantations in areas of more sandy soil and presettlement jack pine dominance than cones from plantations in areas of less sandy soil and presettlement red pine dominance. The numbers of conidia and frequencies of cultural detection of Diplodia species from asymptomatic shoots at a site were positively correlated. These results provide evidence for site-related influences on abundance of pathogen inoculum and asymptomatic persistence on or in red pine crowns that may contribute to differences in frequency and severity of damage from Diplodia shoot blight.     

Longevity of inoculum production by Diplodia pinea on red pine cones

In summer of 2004, pycnidia of Diplodia pinea were observed on cones of Pinus resinosa that had matured and opened during previous years, but had been retained in canopies of trees at a mature red pine plantation in southern Wisconsin. Surveys during the winter and early summer of three consecutive years (2005–2007) to determine incidence and abundance of D. pinea conidia on cones of different ages in this stand. Cones from each age class consistently bore pycnidia with conidia of D. pinea. Although cones collected in June of the year after their maturation tended to yield more D. pinea conidia than older cones, large numbers of conidia were obtained from cones even 3 years after maturation. Perennial availability of inoculum due to persistence of D. pinea on cones of several ages in the overstory or in adjacent stands should be considered when regenerating red pine in areas where this pathogen is known to be present.     

Feeding activities of Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe) as affected by moisture content of wood

The relationship between the moisture content (MC) of wood blocks and the feeding activities of two Japanese subterranean termites, Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe), was investigated with two choice tests. When three wood blocks with low MC (6%–12%), middle MC (79%–103%), and high MC (140%–182%) were exposed to workers of C. formosanus and R. speratus in a choice test, the feeding preferences were found in the middle MC blocks for C. formosanus, and the middle and high MC blocks for R. speratus, although wood blocks of low MC were also attacked. In a second choice test, wood blocks consisting of five pieces with water-impregnated (MC: 133%–191%) top, middle, or bottom piece were exposed to workers of both species. Higher consumptions were generally obtained in water-impregnated wood pieces and bottom pieces.     

Leptoglossus occidentalis and Diplodia pinea: a new insect‐fungus association in Mediterranean forests

Leptoglossus occidentalis, an insect native to North America, was inadvertently introduced into Italy about 1999. The insect damages the cones of conifer trees, especially Pinus pinea (Italian Stone pine). Pinus pinea is also affected by Diplodia pinea, a fungus native to Italy, which is becoming an increasing threat because pine trees are becoming more susceptible to it as a consequence of global warming. Because the insect and the fungus both have the pine cones as a common habitat, a possible interaction between them has been postulated. The aim of this study was to ascertain whether L. occidentalis and D. pinea interact on P. pinea cones. The interaction was studied using real‐time PCR on a group of naturally infected insects collected from a forest, and a group raised in the laboratory and artificially inoculated with D. pinea conidia. Molecular analysis showed that D. pinea DNA occurred on both naturally infected and inoculated insects, but with significant differences between the two groups. The rapid and sensitive molecular technique made it possible to detect D. pinea DNA on the bodies of the insects, and to show that the native D. pinea occurred on the exotic insect.     

Risk from Sirococcus conigenus to understory red pine seedlings in northern Wisconsin

Interest in development of multicohort stands of red pine (Pinus resinosa) in the Great Lakes region of North America prompted an investigation of the potential impact of the shoot blight pathogen Sirococcus conigenus (syn. S. strobilinus) on understory red pine seedlings. In May 2002 and 2003 healthy, 1‐year‐old red pine seedlings were planted in the understory of a maturing red pine plantation in northern Wisconsin in an area with a history of presence of this pathogen. Occurrence of shoot blight symptoms was recorded periodically during the summer, and in each year seedlings were harvested in fall and examined for signs of shoot blight pathogens. By fall 2002 and 2003, respectively, shoot blight incidence was 89% and 98% and most seedlings were dying. Pycnidia with conidia of S. conigenus were present on almost all of the symptomatic seedlings. The conifer shoot blight and canker pathogen Diplodia pinea (syn. Sphaeropsis sapinea) was also detected, though less frequently. Pycnidia of S. conigenus tended to be found more frequently on symptomatic current year's shoots than symptomatic previous year's shoots; the opposite was true for pycnidia of D. pinea. Risk from S. conigenus to understory red pine seedlings should be considered in any plans for development of multicohort red pine stands in areas where the pathogen is present.     

Biological control of Heterobasidion annosum on Pinus pinea by Phlebiopsis gigantea

The efficacy of a native isolate of Phlebiopsis gigantea in protecting the stumps of Pinus pinea against spore infection by Heterobasidion annosum s.str. was investigated. In preliminary tests carried out in stem pieces of P. pinea in confined environment, the efficacy of the isolate was compared with Rotstop^®, the commercial formulation prepared from a north European P. gigantea strain. Both showed a fully protective effect against artificial inoculation of H. annosum. Moreover, in stump treatment experiments carried out in a coastal P. pinea forest close to Rome, the native P. gigantea was effective against heavy airborne inoculum of H. annosum. Some natural infection by P. gigantea occurred in the stumps, but it was unable to control the pathogen. Random amplified microsatellite analysis allowed to recognize the presence of different P. gigantea strains in the forest.     

Diplodia shoot blight and asymptomatic persistence of Diplodia pinea on or in stems of jack pine nursery seedlings

Diplodia pinea (syn. Sphaeropsis sapinea) is known as a major cause of damage to red pine (Pinus resinosa) seedlings in nurseries. The fungus can also be a latent pathogen of red pine seedlings, persisting in the absence of gross symptoms and later proliferating under conditions that induce host stress. In the fall of 2004, three nurseries in Wisconsin were surveyed to determine the potential for the occurrence of Diplodia shoot blight on jack pine (Pinus banksiana) seedlings and the persistence of D. pinea on or in asymptomatic seedlings of this species. Incidence of shoot blight was quantified in five 1 m long segments of an interior row in each of two survey areas in each nursery. The pathogen was identified on symptomatic seedlings collected in these areas on the basis of presence of characteristic pycnidia and conidia. Five groups of 20 asymptomatic seedlings were also collected in each of the two survey areas in each nursery. A segment of the lower stem/root collar from each of these asymptomatic seedlings was surface‐disinfested and culturally assayed using tannic acid agar. The mean incidence of shoot blight (as high as 9%) and mean frequency of cultural detection from asymptomatic seedlings (as high as 20%) were greatest in proximity to red pine windbreaks which are a source of inoculum. Only D. pinea was confirmed from subsets of symptomatic and asymptomatic seedlings which were tested using mt SSU rDNA polymerase chain reaction (PCR) primers that allow differentiation of D. pinea from D. scrobiculata and other fungi in the genus Botryosphaeria and related anamorphic fungi. Jack pine seedlings inoculated with D. pinea isolates obtained from asymptomatic nursery seedlings developed shoot blight symptoms in greenhouse trials. Thus, the ability of D. pinea to damage jack pine seedlings in nurseries has been documented and the potential for virulent strains of this latent pathogen to be distributed on asymptomatic jack pine seedlings from nurseries has been confirmed.     

Associations between Tomicus destruens and Leptographium spp. in Pinus pinea and P. pinaster stands in Tuscany,central Italy

The association between Tomicus destruens and fungi of the genus Leptographium was studied in Pinus pinea and P. pinaster forests in Tuscany, central Italy. Fungi were isolated from adult beetles and from pine tissues from infested trees. On average, Leptographium spp. were associated with 18% of beetles in breeding galleries, 35% of emergent brood beetles and 18% of beetles undergoing maturation feeding in pine twigs. The fungal species most frequently identified were Leptographium wingfieldii and L. lundbergii while L. guttulatum and L. serpens were also found.     

Biocontrol of decay in seasoning utility poles. I. Growth rate and colonizing ability of bluestain and decay fungi in vivo and in vitro

Seasoning (air drying) of utility poles for 6–12 months is essential before preservative treatment can be achieved. However, during seasoning, pine sapwood is often colonized by decay fungi, thereby compromising the performance and service life of the poles. This study investigated the potential of bluestain fungi to act as short‐term biocontrol agents against decay during seasoning. An important attribute for biocontrol is rapid growth, so growth rates of common bluestain (Ceratocystis coerulescens, Ophiostoma minus, Ophiostoma piceae, Ophiostoma piliferum, Sphaeropsis sapinea) and decay fungi (Heterobasidion annosum, Phlebiopsis gigantea, Stereum sanguinolentum) were compared on agar medium and pine in logs at various temperatures. On agar, the growth temperature optimum of most bluestain fungi and all the decay fungi was ～25°C, with little growth at ≤5°C or above 32.5°C. Overall, the fastest growing were S. sapinea and O. minus. In logs, the most effective colonizers were S. sapinea and O. minus with pathogenic abilities that made them well fitted to colonize the sapwood of freshly felled pine. Within these species, certain isolates produced much larger lesions in phloem and the sapwood tangential plane than all the decay fungi. Notably, there was significant variation in colonizing ability between different isolates within a species, emphasizing the need for testing a range of isolates when selecting a potential biocontrol agent.     

卵巢线虫(Contortylenchus genitalicola)在中国松褐天牛上的分布与作用研究

[目的]日本学者Kosaka与Ogura发现松褐天牛成虫除携带松材线虫外,雌成虫卵巢内还携带有另一种线虫,他们将其命名为卵巢线虫,并认为这种线虫是松褐天牛成虫的寄生性线虫。我国对该种线虫的研究迄今未见报道。为了证明这种线虫在我国是否存在和分布,开展了本项研究。[方法]分期分批捕获刚羽化的松褐天牛雌雄成虫,采用解剖松褐天牛成虫松树木质部及感病的松褐天牛幼虫等方法,调查卵巢线虫的存在与分布。[结果]通过调查研究,发现我国的松褐天牛成虫体内有该卵巢线虫存在,分布于松褐天牛成虫、幼虫体内和松树木质部3个部位;在松褐天牛成虫体内的卵巢线虫通过松褐天牛雌成虫产卵而接种、进入寄主树木木质部中。同时,木质部的卵巢线虫也有一部分进入松褐天牛幼虫体内寄生,另一部分仍在木质部生活,当松褐天牛幼虫再次发育为成虫时,在木质部中的卵巢线虫和已被寄生的松褐天牛幼虫体内的卵巢线虫再次进入松褐天牛成虫体内,完成循环。卵巢线虫在松褐天牛雌、雄虫体内均有分布,携带率为44.4%,其中松褐天牛雌成虫携带率为43.8%,松褐天牛雄成虫携带率为45.0%,两者间无显著差异;每头松褐天牛成虫平均携带卵巢线虫574条,其中雌成虫平均携带816条,雄成虫平均携带308条,具显著差异。初步研究表明,卵巢线虫在松褐天牛成虫体内只能完成产卵到1~4龄的幼虫阶段,不能完成一个完整的世代;在松褐天牛幼虫体内寄生和在木质部生活的卵巢线虫能完成一个完整世代,但具体过程尚不清楚;调查中未观察到卵巢线虫对松褐天牛成虫有寄生致病或致死的现象,但对松褐天牛幼虫有寄生致死的能力;卵巢线虫常与松材线虫同时存在,而且侵入松树及离开松树的方式与松材线虫相同。[结论]我国松褐天牛体内也发现有卵巢线虫存在;目前尚不能证明该线虫对松褐天牛成虫具有寄生致死性,但对松褐天牛幼虫具有一定的寄生致死性;卵巢线虫的生活史与松材线虫相似,是否与松材线虫一样对松树具有危害性以及其病理作用还有待于进一步研究。     

Effects of oleoresins and monoterpenes on in vitro growth of fungi associated with pine decline in the Southern United States

As a means of exploring pine resistance to root disease and declines, the effects of host plant secondary metabolites on the growth of root colonizing fungi associated with three diseases/declines of southern pines – loblolly pine decline, littleleaf disease and annosum root rot were tested. The associated fungi –Leptographium huntii, L. serpens, L. terebrantis, L. procerum, Heterobasidion annosum and Phytophthora cinnamomi– were grown in saturated atmospheres or in direct contact with, pure monoterpenes and crude oleoresin collected from the four southern pines (Pinus taeda, P. eschinata, P. palustris and P. elliotti) for 7 day. Fungal growth was measured at 3, 5 and 7 day. Root‐infecting fungi differed significantly in sensitivity to crude oleoresin and pure monoterpenes. All fungi tested were inhibited, to some extent, by the resins tested. H. annosum and P. cinnamomi were strongly inhibited by all the monoterpenes tested. The ophiostomatoid fungi were significantly less affected by the compounds tested. L. huntii and L. serpens were less inhibited by monoterpenes than either L. terebrantis or L. procerum. These fungal growth studies show that the kind and amount of secondary metabolite produced by the host plant have a profound effect on tree pathogens. Alterations of tree physiology may have implications for defenses against tree pathogens as well as to the ecology and management of forest ecosystems. Difference in incidence of root disease observed in the field may be explained by the ability of the fungus to tolerate these host defense mechanisms.     

Detection of Diplodia pinea in asymptomatic pine shoots and its relation to the Normalized Insolation index

Diplodia pinea (syn. Sphaeropsis sapinea), a common pathogenic fungus, causes considerable damage in Italy, particularly to pine stands in which trees are subjected to environmental stress. The occurrence of D. pinea in symptomless Pinus nigra shoots was investigated and related to the amount of radiation received by the trees growing on a site in a year, expressed as the Normalized Insolation index (NIi). Twenty‐seven pines were selected from nine locations in Trentino (northern Italy). For each pine the incidence of the fungus in apparently healthy shoots was determined by both culturing on an agar medium and application of real‐time PCR. The incidence of D. pinea determined by culturing samples taken from asymptomatic trees was 59% (16 of 27 trees), compared with 85% found using real‐time PCR (23 of 27 trees). Detection of the pathogen in healthy pine tissue was positively correlated (p < 0.05) with the NIi values, using both detection methods.     

马尾松不同种源氨基酸含量与抗松材线虫病的关系

在选出和验证抗病马尾松种源的基础上,研究了马尾松不同抗病性种源的树体内氨基酸含量的差异。结果表明,抗病种源在接种松材线虫前后均比较感种源含有较少的组氨酸、苏氨酸、甘氨酸、异亮氨酸、胱氨酸、甲硫氨酸、亮氨酸、酪氨酸和精氨酸等９种游离氨基酸。它们与感病指数均呈正相关,这说明游离氨基酸含量低的马尾松种源抗松材线虫病。１１种固态氨基酸的含量与感病指数有负相关性,说明其含量较高的种源具较强抗病性能。     

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.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

松材线虫(Bursaphelenchus xylophilus)耐久型幼虫(LIV)携带的香茅醇假单胞杆菌(Pseudomonas citronellolis)分离与鉴定

[目的]目前有关松材线虫与伴生细菌的关系及伴生细菌的病原作用是松树枯萎病研究的重点。为了揭示松材线虫与伴生细菌之间存在的密切关系,作者对松材线虫L_IV幼虫携带的细菌进行了分离鉴定。[方法]根据培养性状和16S rDNA序列同源性以及系统发育学等方面进行分析鉴定。[结果]确定L_IV幼虫携带的是香茅醇假单胞杆菌（Pseudomonas citronellolis）,携带率为100%;每条L_IV幼虫携带量在1.4×10⁵~4.5×10⁵。L_IV幼虫生活在松褐天牛体内,是引起松材线虫病侵染流行的唯一虫态;新发现的香茅醇假单胞杆菌能分解纤维素及降解或合成萜烯和酚类化合物。[结论]L_IV幼虫携带香茅醇假单胞杆菌的发现,揭示了松树、松褐天牛、松材线虫、细菌同为一体的紧密关系,并为揭示松树枯萎病机制提供了一种新病原和重要的研究思路。     

Seasonal dynamics of wood formation: a comparison between pinning,microcoring and dendrometer measurements

Three different methods were evaluated for analysing wood formation of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus sylvestris L.) in Finland. During two growing seasons, wood formation dynamics were determined both by wounding the cambium with a needle followed by localisation of the wound-associated tissue modification after the growing season (pinning), and by extracting small increment cores during the growing season (microcoring). Stem radius was additionally monitored with band dendrometers. For Norway spruce, pinning and microcoring yielded similar dates for the onset of wood formation. The timing of wood production during the growing season was also similar for pinning and microcoring. For Scots pine, the onset of wood formation was recorded from microcores almost 2 weeks later than from pinning samples. In Scots pine, microcore measurements also produced somewhat later cessation dates for tracheid formation than the pinning samples. For both tree species, the total number of tracheids formed during the growing season was, however, about the same for pinning and microcoring. Dendrometer results clearly differed from those of pinning and microcoring. In particular, the dendrometers showed an increase of stem radius considerably earlier in spring, when the other methods did not detect wood formation. Thus, pinning and microcoring currently represent the most reliable techniques for detailed monitoring of wood formation.     

Fallen wood decomposition ofPinus koraiensis andTilia amurensis

Fallen wood decomposition rate ofPinus koraiensis andTilia amurensis in broadleaved Korean pine forest was studied in this paper. The result showed that decomposition rate of fallen wood was different from that of little diameter wood and coarse woody debris for the same tree species. Fallen wood decomposition was generally rotten from outside to inside. And decomposition speed of fallen woods was different according to tree species and site, and it was also related to diameter of fallen woods. Decomposition depth ofTilia amurensis fallen wood for 17 years was 14 cm, but that ofPinus koraiensis in the same condition was less than 7 cm.Tilia amurensis was decomposed faster thanPinus koraiensis. For same tree species, if the diameter was small, the decomposition speed was quick. This project was supported by Natural Science Foundation of China (No. 39670144) and funded by the Opened Research Station of Changhai Mountain Forest Ecosystem, Chinese Academy of Sciences. (Responsible editor: Zhu Hong)