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.     

The aggressiveness of Sphaeropsis sapinea on Austrian pine varies with isolate group and site of infection

The aggressiveness of the A and B isolate groups (morphotypes) of Sphaeropsis sapinea on shoot tips and lower stems of Austrian pine (Pinus nigra) was compared. The distinct differences in aggressiveness between the two S. sapinea groups, previously reported for other conifers, are confirmed for Austrian pine. However, the relative aggressiveness of the B group isolate varied by site of infection. Although the B group isolate of S. sapinea was not aggressive on shoot tips of Austrian pine, it was aggressive on stems of this host.     

Surveys for asymptomatic persistence of Sphaeropsis sapinea on or in stems of red pine seedlings from seven Great Lakes region nurseries

The fungus Sphaeropsis sapinea persists on or in stems of asymptomatic red pine (Pinus resinosa) nursery seedlings, and proliferates to cause collar rot and mortality after planting. In the spring of 2002, seven nurseries were surveyed to determine the potential range in frequency of asymptomatic persistence: three operated by Wisconsin Department of Natural Resources (DNR), two by Minnesota DNR, one by Michigan DNR, and one by USDA Forest Service (in Michigan). At each nursery five groups of 20 asymptomatic red pine seedlings were collected near an inoculum source (red pine windbreak), if present, and five groups of 20 asymptomatic seedlings were collected away from such a source (1400 seedlings total). A segment of the lower stem/root collar from each seedling was surface disinfested and incubated on tannic acid agar. Transfers were made from resulting colonies and the pathogen identified from pycnidia and conidia produced in culture. The pathogen was identified from asymptomatic seedlings collected in all Wisconsin and Minnesota nurseries, but was never detected from seedlings from the Michigan DNR or USDA Forest Service nurseries. Frequencies of detection were greater (as high as 88%) from asymptomatic seedlings near red pine windbreaks including diseased trees than from seedlings distant from such windbreaks. A subset of isolates from asymptomatic seedlings was characterized using inter‐simple sequence repeat–polymerase chain reaction analysis. Most isolates were the A group of S. sapinea, but B group isolates (recently named Diplodia scrobiculata) were also obtained from one nursery. One Minnesota nursery was more extensively sampled in 2003, with 17–44 groups of five asymptomatic red pine seedlings collected in four separate fields (525 seedlings total). The mean frequency of detection of the pathogen in these four fields ranged from 40 to 71%. Persistence of S. sapinea on or in asymptomatic seedlings continues to be problematic, not only because of the potential for subsequent seedling mortality, but also as a means by which a pathogen may be widely distributed.     

Pre‐ and post‐inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings

In order to assess the influence of water stress on the development of Sphaeropsis sapinea cankers in Pinus halepensis, the stems of 4‐ to 5‐year‐old potted seedlings were artificially inoculated with the fungus before and after being kept at controlled water regimes from April 1997 to March 1998. In the pre‐water‐stress inoculation experiment, the canker length, measured 5 months after inoculation (September 1997), was greater in seedlings predisposed to extreme water deficit (midday needle water potential between ?4.5 and ?5.5 MPa). In the post‐water‐stress inoculation experiment, the fungus was inoculated in April 1998, after irrigation had enabled the seedlings to resume normal needle water potential. In this case also, at 5 months after inoculation, longer cankers were visible in seedlings that had been subjected to extreme water deficit. These findings suggest that the occurrence of marked water stress, although apparently tolerated by Aleppo pine, can enhance the development of S. sapinea cankers in this species, regardless of whether the stress occurs before or after infection by the fungus.     

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。     

Sphaeropsis shoot blight and altered nutrition in red pine plantations treated with paper mill waste sludge

During 1993 progressive, severe shoot blight and canker disease in crowns of mature, merchantable red pines (Pinus resinosa) in central Wisconsin was noted in plantations in which paper mill waste sludge previously had been applied. For eight treated plantations and six non‐treated plantations, incidence of shoot blight attributed to the pathogenic fungus Sphaeropsis sapinea (syn. Diplodia pinea) was quantified during 1993 or 1994. Foliage and soil samples also were collected for analyses. Sphaeropsis shoot blight was more frequent in treated plantations than in the non‐treated stands (means of 81% of trees and 10.2% of shoots compared with 7% of trees and 0.1% of shoots, respectively). Consistent with other reports of damage caused by some diseases of conifers in situations of altered host nutrition, mean foliar N concentrations were higher in treated plantations (1.61%) than non‐treated plantations (1.31%) (p < 0.001). Mean foliar Zn, Mn, Cu, and Al concentrations were lower in treated plantations than non‐treated plantations, and mean soil P, Ca, and Mg concentrations were higher in treated plantations than non‐treated plantations.     

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.     

松球壳孢菌的形态学与18S rDNA ITS序列比较分析

以研究东北三省樟子松球壳孢菌Sphaeropsis sapinea的遗传多样性为目的,自黑龙江、吉林、辽宁省不同地区收集、分离获得樟子松球壳孢菌45个菌株,寄主均为樟子松;收集美国菌株2个,分别分离自脂松、班克松.运用形态学和分子生物学手段比较47个菌株的形态学及18S rDNAITS序列.结果表明:(1)所有47个菌株在形态上没有明显差别;(2)除美国菌株STBl外,其他46个菌株在培养特性上没有明显差别;(3)东北三省的45个菌株之间的遗传距离均小于0.09,与寄主为脂松的A型菌株(STAl)亲缘关系较近,相似度高于98%,与寄主为班克松的B型菌株(STBl)遗传距离为0.13,亲缘关系较远.(4)同一地域、不同地域来源的菌株其ITs序列相同,而地理来源相同的菌株其ITs序列也存在一定的差异;(5)相同寄主上的菌株其ITs序列存在差异,而不同寄主上的菌株其ITS序列相同.综上得出以下结论:东北三省不同地区樟子松上的45个松球壳孢菌菌株为A型.球壳孢菌菌株之间的遗传差异与菌株的寄主及地域性没有明显的关系.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

Organ-dependent induction of systemic resistance and systemic susceptibility in Pinus nigra inoculated with Sphaeropsis sapinea and Diplodia scrobiculata

Systemic induced resistance (SIR) is a well-known host defense mechanism against pathogen attack in herbaceous plants, but SIR has only recently been documented in conifers. We tested if inoculation of Austrian pine (Pinus nigra Arnold) with Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton or Diplodia scrobiculata de Wet, Slippers and Wingfield results in SIR or systemic induced susceptibility (SIS) to subsequent colonization by S. sapinea. Induction at the stem base resulted in significant (P < 0.01) SIR in the upper stem, and induction in the upper stem resulted in significant (P < 0.05) SIR at the stem base, indicating that SIR is bidirectional in Austrian pine. However, inoculation at the stem base resulted in significant (P < 0.01) SIS in shoot tips, demonstrating that, in the same host species, the expression of resistance can be organ-dependent, resulting in either SIR or SIS depending on the site of challenge infection. Systemic induced resistance in the stem was associated with induced lignification, supporting a potential role for this defense mechanism in disease resistance. Systemic induced susceptibility has been documented before, but this is the first demonstration of organ-dependent expression of both SIR and SIS in a tree or any other plant.     

Induction of phenylalanine ammonia lyase activity and control of Sphaeropsis sapinea infection in Pinus radiata by 5-chlorosalicylic acid

Phenylalanine ammonia lyase (PAL) activity increased rapidly in Pinus radiata seedlings after treatment with 2 mm 5-chlorosalicylic acid (5CSA) and peaked after 48 h at four-times the level of the control. Elevation of PAF is often associated with the induction of host resistance mechanisms. Thus, effects of 5CSA on subsequent infection of P. radiata by Sphaeropsis sapinea was investigated. Only one out of 16 seedlings treated with 2 mm 5CSA, 4 days before inoculation, developed deadtop compared to 14 of the controls. The 5CSA was directly inhibitory at this concentration and reduced growth rate of S. sapinea on agar by 27%. However, antifungal activity does not fully account for the observed disease control afforded by 5CSA since 0.5 mm and 0.25 mm 5CSA, which did not inhibit growth of S. sapinea, significantly reduced the incidence of deadtop. Furthermore, 1 mm 5CSA protected seedlings against fresh inoculation challenge for up to 32 days after application. Such durable resistance is unlikely to be attributable to the direct effects of the applied compound and may suggest the involvement of inducible host resistance mechanisms.     

Pathogenicity of A and B morphotypes of Sphaeropsis sapinea confirmed on American larch (tamarack) and European larch

Fungi identified as Sphaeropsis sapinea (or synonyms) have been reported from Larix. Lack of pathogenicity tests, confusion about the identity of isolates mentioned in previous literature, and existence of distinct populations (A and B morphotypes) of the pathogen indicated the need to evaluate the ability of S. sapinea to cause disease of larch. Elongating snoot tips of Larix decidua, Larix laricina, and known hosts of the pathogen, Pinus banksiana and Pinus resinosa, were inoculated with water-agar plugs colonized with an A or a B isolate, or sterile plugs (controls). Each of five replications included five seedlings for each treatment-species combination. After 6 weeks, no symptoms had developed on control seedlings, but two-way analyses of variance revealed significant effects of isolate morpnotype and host on both incidence and severity of disease (values of p ≤ 0.01). The A isolate killed almost all shoot tips, but the B isolate killed from no P. resinosa shoots to 56% of L. laricina shoots. The average length of shoot killed by the A isolate was also greater than that killed by the B isolate. Response to the B isolate again varied among species, with greater average lengths of shoot killed on the larches (compared with the pines). Both A and B morphotypes of 5. sapinea should be considered among the fungi encountered on L. decidua and L. laricina.     

The pine pathogen Diplodia sapinea is associated with the death of large Douglas fir trees

Climate change poses severe pressures to European conifer forests. Using non-native tree species, such as Douglas fir (Pseudotsuga menziesii), is one proposed strategy to circumvent adverse effects for forest management. However, novel forest health risks can impair the cultivation of non-native trees. In 2022, we observed large Douglas fir trees (approximately 40–50 years old, diameter at breast height (dbh) 21–41 cm) that had recently died in spring or summer 2022 in three forest stands in Eastern Austria. Intensive resin flow, blue-staining of the sapwood and the absence of bark- and wood-boring insects indicated a fungal infection. Isolations from blue-stained sapwood of the dead trees consistently yielded cultures of the opportunistic pathogen Diplodia sapinea. In a greenhouse wound inoculation experiment, seven D. sapinea isolates obtained from Douglas fir caused phloem necrosis, blue-staining of sapwood and mortality and thus displayed pathogenicity towards seedlings of both Ps. menziesii and its common host, Scots pine (Pinus sylvestris). Diplodia sapinea produced significantly longer areas of blue-stain as well as higher and faster mortality in Ps. menziesii compared to P. sylvestris. We conclude that D. sapinea substantially contributed to the death of seven of the 13 examined large Douglas fir trees. While this fungus has been described as a pathogen of young Douglas fir trees before, this is the first report that it can potentially kill large individuals of this conifer species under drought conditions. Thus, our results indicate that D. sapinea could represent a severe threat to the cultivation of Ps. menziesii in European forestry.     

Excised shoots of top-pruned red pine seedlings,a source of inoculum of the Diplodia blight pathogen

Diplodia pinea causes shoot blight and collar rot diseases of pines in forest tree nurseries and sporulates on colonized seedling needles and stems. In late summer 2005, pycnidia of D. pinea were observed on shoots that had been excised by top pruning red pine seedlings earlier that summer during the third season of growth. This observation prompted surveys to determine the incidence and abundance of D. pinea conidia on excised shoots. At each of two nurseries, excised shoots were collected from the seedling canopy and adjacent alleyway soil in two subplots in each of five beds (plots). Excised shoots from both nurseries bore pycnidia with conidia of D. pinea. A water washing and filtration technique was used to quantify D. pinea conidia extracted from these shoots. Excised shoots collected from the seedling canopy yielded more D. pinea conidia than shoots collected from adjacent alleyway soil. Collection and removal of excised shoots resulting from top pruning of pine nursery seedlings should be considered as a means of reducing inoculum in areas where D. pinea is present.     

不同生物控制技术对樟子松林分生长及松枯梢病发病程度的影响

通过对樟子松林分进行生物控制技术的研究可知,在中龄林进行施用菌剂、切根、人工整枝试验对促进樟子松树势的恢复、树木胸径生长、增强树势等方面等有明显作用:在胸径生长方面,3个处理与对照差异显著,其中切根5年后的樟子松林分胸径生长高于施用菌剂和人工整枝处理,年生长量达1.89cm;3个处理与对照相比,大大减少了松枯梢病发病机率,其中修枝处理防控病害的效果最好。     

Effect of damaged pine needles on growth and development of pine caterpillar larvae

Chinese pine caterpillar (Dendrolimus tabulaeformis) larvae were fed with pine needles of different degrees of damage to evaluate the effects of pine needles on the growth and development of larvae. The results showed that the nutritional index of the larvae declines after feeding on the damaged pine needlings. The lowest amount of food ingested and voided feces, the lowest nutritional index, slowest development, lightest pupae and most mortality were found in those pine caterpillar larvae fed with pine needles which were 50% damaged. The damaged pine needles significantly affected the population dynamics of Chinese pine caterpillars. The nutritional indices of larvae fed with 25% and 75% damaged pine needles were similar. The nutritional index of the dark morphs was higher than that of the tinted morphs, however, their mortality was lower than that of the tinted morphs. This phenomenon was reversed at the later stage of development when the larvae were fed on 50% damaged pine needles. __________ Translated from Journal of Beijing Forestry University, 2005, 27(6): 83–88 [译自: 北京林业大学学报, 2005, 27(6): 83–88]     

毒蘑菇菌株及毒素粗提液对樟子松枯梢病菌生长的影响

厚环鹅膏、鹅膏菌、珊瑚菌、白霜杯伞、冠状柄环菇、细鳞环柄菇、鳞柄白鹅膏、绒白乳菇、毒蝇鹅膏9个毒蘑菇菌株及毒素粗提液对樟子松枯梢病原菌菌丝生长影响的研究结果表明:除厚环鹅膏和鹅膏菌外,其他7个菌株对病原菌生长均有一定的抑制作用;鳞柄白鹅膏和细鳞环柄菇相对抑制效果最好,分别为10.80和10.40。除毒蝇鹅膏外,其他8个菌株的毒素粗提液对病原菌菌丝生长均有一定的抑制作用,珊瑚菌和绒白乳菇毒素粗提液的抑菌效果最好,抑菌率分别达到63.89%和58.89%。     

PCR primers for identification of Sirococcus conigenus and S. tsugae,and detection of S. conigenus from symptomatic and asymptomatic red pine shoots

Regions of diversity in the internal transcribed spacer (ITS) sequences of Sirococcus species were exploited to design primer pairs used in a PCR-based method for the identification of the conifer shoot blight pathogen Sirococcus conigenus and the closely related fungus Sirococcus tsugae. The specificity of each primer pair for the respective fungus, detection limits and utility for detection from host material were confirmed. The S. conigenus primers were then used to detect this pathogen in tissues of symptomatic or apparently healthy red pine shoots collected at six locations in Wisconsin and Michigan and results compared with those obtained using a cultural assay. For needles, bark and wood of symptomatic shoots, the mean frequencies of detection of S. conigenus using the PCR-based methods were consistent (≥7.5 out of 10) and always greater than for the cultural assay. Detection from symptomatic shoots using the cultural assay was more frequent from needles than from bark or wood. Both the PCR-based method and the cultural assay detected S. conigenus in similar frequencies from asymptomatic shoots, although less frequently than from symptomatic shoots. The efficiency of the PCR-based method and its utility for direct testing of host material should make it particularly useful in areas where multiple shoot blight pathogens are found.     

A comparison of needle retention on Japanese black pine and Japanese red pine

Needle retention (number of needle sets), needle density, height increment and radial increment were surveyed on seven Japanese black pines (Pinus thunbergii Parl.) and seven Japanese red pines (Pinus densiflora Sieb. et Zucc.) growing on the same site. Number of needle sets on branches with respect to whorl position was estimated visually. In 1999, maximum summer needle retention was observed on the fifth and sixth whorl from the top, with values of 3.4 needle sets for the black pines and 2.4 needle sets for the red pines, respectively. The needle trace method (NTM) was used to determine needle retention and needle density along the main stems retrospectively for the years 1968–1998. The long-term mean summer needle retention along the main stem was 3.7 needle sets for the black pines and 2.2 needle sets for the red pines. In both pine species, the number of needle sets varied from year to year. However, the long-term budget between newly born and annually shed needle sets was in equilibrium. The long-term average of needle density per cm of stem shoot was 9.4 needle pairs for the black pines and 7.4 needle pairs for the red pines. The results showed clear intra-specific and inter-specific similarities in needle retention, height increment and radial increment trends. The values for number of needle sets, height increment and radial increment positively increased with favorable growing conditions, whereas the value of needle density had an opposite tendency.