Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine

ABSTRACT The aggressiveness of Sphaeropsis sapinea isolates was compared on water-stressed and nonstressed 3-year-old red pines (Pinus resinosa) in greenhouse and growth chamber experiments. Water was withheld from stressed seedlings to achieve mean predawn needle water potentials (psi(PD)) above -1.9 MPa. The lowest mean psi(PD) of well-watered seedlings was maintained at or above -0.8 MPa. Young shoots were inoculated by placing colonized agar plugs on wounds made by removing a needle fascicle. Two isolates of each recognized morphotype (A and B) were used in the greenhouse experiment and two isolates of morphotype A were used in the growth chamber experiment. After 4 weeks, isolates of morphotype A caused more severe symptoms and could be recovered farther from the inoculation site on water-stressed than on nonstressed trees in both experiments. In the greenhouse experiment, isolates of mor-photype A also caused more severe symptoms and could be recovered farther from the inoculation site than isolates of morphotype B, regardless of watering regime. These results indicate that water stress at levels observed typically in the field can result in increased disease development by isolates of S. sapinea morphotype A on red pine. The reduction of water stress of red pines in the field may reduce losses due to Sphaeropsis shoot blight.     

Sphaeropsis sapinea and Water Stress in a Red Pine Plantation in Central Wisconsin

ABSTRACT A study was conducted to determine the effects of water stress resulting from competing vegetation on disease development of Sphaeropsis sapinea in red pine plantations. A 9-year-old plantation was selected in 1992 and experiments were conducted for three consecutive years. Four treatments were assigned at random to individual trees: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. Two isolates of each S. sapinea morphotype (A and B) were used to inoculate wounded lateral shoots. Disease development was measured as the maximum distance below the inoculation site at which necrotic needles were observed. Nonwatered trees with competing vegetation (nontreated condition) had significantly lower predawn needle water potentials (more water stress) and more severe disease development than trees that received the herbicide, water, or combined herbicide and water treatments. The most severe disease occurred in the driest year and the least in the wettest year. Competing vegetation indirectly affected disease development by inducing water stress, even in relatively moist years, on trees previously considered well established. Isolates of morphotype A were more aggressive than isolates of morphotype B. Conclusions from this research have implications for sustainable management of the region's conifer forests.     

Differential Inhibition of Sphaeropsis sapinea Morphotypes by a Phenolic Compound and Several Monoterpenes of Red Pine

ABSTRACT The in vitro effects of a red pine phenolic compound (pinosylvin), a phenolic compound common to other species (tannic acid), and the major red pine monoterpenes (alpha-pinene, beta-pinene, and delta-3-carene) on spore germination and mycelial growth of Sphaeropsis sapinea were examined. Two A and two B morphotype isolates were used. At 88 mug/mm(2), pinosylvin inhibited spore germination of all four isolates (98 to 100%). At 8.8mug/mm(2), spore germination of B isolates was inhibited more than that of A isolates (73 versus 30%). Pinosylvin also inhibited mycelial growth of B isolates more than that of A isolates (84 versus 13% at 88 mug/mm(2)). Tannic acid stimulated or had little affect on spore germination and had little affect on mycelial growth of either morphotype. Spore germination of B isolates was inhibited more than that of A isolates by beta-pinene at saturation (79 versus 37%). Spore germination of B isolates was inhibited and germination of A isolates was stimulated by delta-3-carene below saturation (49 versus -7%). Mycelial growth of B isolates was inhibited more than that of A isolates by all monoterpenes at saturation. Differences observed between morphotypes below saturation were significant only for beta-pinene. These results demonstrate the biological activity of a phenolic compound and monoterpenes that occur in red pine. The differential responses might provide means of distinguishing morphotypes and offer a potential explanation for ecological specialization.     

Further studies on the role of monoterpenes in pine host selection and oviposition ofThaumetopoea pityocampa

In a young mixed pine plantation (Pinus nigra andP. pinea) in central Italy, the influence of the pine needle monoterpenes (R)-(+)-limonene, (S)-(−)-limonene and (1S)-(−)-β-pinene on the oviposition activity of the pine processionary moth,Thaumetopoea pityocampa (Denis et Schiffermüller), was assessed. Two application methods were compared: spraying the trees with the substances emulsified with water, and setting up polyethylene dispensers from which the monoterpenes evaporated. During the moth’s flight period weekly treatments (at 500 mg of monoterpene /l water) were carried out and the evaporation from the polyethylene dispensers (each with 5 g of a single monoterpene) was checked on each occasion by weighing the dispensers. The GLM analysis did not uncover any difference between the methods of application or between the pine species.T. pityocampa laid a significantly (P=0.01) lower number of egg masses on the trees treated with (1S)-(−)-β-pinene than on those treated with the other substances and on the control trees. A ∼45% reduction in egg mass number was obtained on the pines treated with (1S)-(−)-β-pinene, independent of the level of infestation, method of application or pine species. (R)-(+)-limonene and (S)-(−)-limonene were not effective. http://www.phytoparasitica.org posting July 29, 2008.     

湿地松针叶中的松脂含量及其单萜,倍半萜的组成与抗褐斑病的关系

 研究分析了湿地松20个抗病无性系和14个易感株针叶中的松脂含量,结果表明抗病湿地松针叶中的松脂含量为7.17%,明显比易感株含量5.46%高。顶冠针叶的松脂含量与侧枝针叶的含量基本相近。对9个抗病无性系和4个易感株针叶挥发油中的单萜和倍半萜研究表明,湿地松针叶中的单萜和倍半萜的种类较多,不同无性系(或单株)间高含量单萜和倍半萜种类较稳定,某些低含单萜组分与抗病性之间存在着一定的联系。抗病无性系针叶中都缺乏在感病针叶中普遍存在的异龙脑和香草醇。     

Effects of Water Stress on Colonization of Poplar Stems and Excised Leaf Disks by Septoria musiva

ABSTRACT Septoria musiva causes leaf spot and canker diseases of trees in the genus Populus, and is one of the most damaging fungal pathogens of hybrid poplar in eastern North America. The effect of host water stress on Septoria canker development was studied in two separate greenhouse experiments. Hybrid poplar clones NM6, NC11396, and NE308 were stressed by withholding water until predawn water potential fell below -1.0 MPa. Stems were treated by removing a leaf and applying agar plugs that were either colonized by S. musiva (inoculated) or sterile (control) to the wound. Cankers on inoculated water-stressed trees were significantly larger than those on nonstressed trees. A leaf disk assay also was conducted three times with the NM6 and NE308 trees. We cut two disks from each of 120 stressed and 120 well-watered trees, placing them on water agar in 24-well tissue culture plates. A conidial suspension was applied to one disk in each pair and sterile water to the other. Inoculated disks from water-stressed trees developed less necrosis than those from well-watered trees. These results demonstrate that environmental influences on host condition must be considered in evaluating resistance of clones proposed for widespread culture of hybrid poplar.     

Detection of Latent Sphaeropsis sapinea Infections in Austrian Pine Tissues Using Nested-Polymerase Chain Reaction

ABSTRACT Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies. The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples. Correlation (chi-square) analyses indicated that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.     

Growth environment and seedling age affect constitutive and inducible defence in radiata pine

There is increasing interest in induced resistance for crop protection. This study investigated effects of growth environment on inducible defences in developing radiata pine (Pinus radiata) seedlings. Seedlings were grown in the presence or absence of Trichoderma in either a temperature-controlled growth room or in a greenhouse. After 3, 5 or 7 months, seedlings were sprayed with 2.25 mm methyl jasmonate (MJ), 1 week before quantification of monoterpenes and total phenolics. The magnitude of induction of monoterpenes and total phenolics by MJ was inversely proportional to constitutive content, was greater in the greenhouse than the growth room and was greater at 3 months than at 7 months. This may indicate a cost-saving strategy to limit defence induction once constitutive defence reaches a certain threshold. Growth room seedlings expressed greater constitutive resistance to terminal crook (Colletotrichum acutatum) and to diplodia dieback (Diplodia sapinea) than greenhouse plants, consistent with the relative differences in constitutive defence chemistry. Indeed, while MJ induced resistance to diplodia dieback in both environments, there was no difference in terminal crook incidence (4.1%) between MJ-treated and untreated seedlings in the growth room. By the end of the study, growth room seedlings had c. 70% more β-pinene in stems and 140% more total phenolics in needles than greenhouse plants but were 40% smaller, indicating a defence–growth trade-off. Trichoderma did not have a significant effect on defence chemistry or on pathogen resistance. These results demonstrate the potential to manipulate defence in radiata pine by modification of the growth environment.     

Genetic variation in tolerance of host monoterpenes in a population of the ascomycete canker pathogen Crumenulopsis sororia

Twenty families of single-ascospore isolates of the canker pathogen Crumenulopsis sororia were collected from eight trees of Pinus sylvestris in a native pinewood. Measurements were made of growth rate of each isolate in the presence and absence of a saturating atmosphere of the five principal monoterpenes of P. sylvestris cortical oleoresin (α-pinene, β-pinene, 3-carene, β-myrcene and limonene).
All monoterpenes significantly reduced the mean linear growth rate of the C. sororia population. However, there was highly significant genetic variation among isolates in their tolerance of all five monoterpenes. The extent of genetic variation was greatest for β-myrcene and limonene tolerance, and least for α-pinene and 3-carene tolerance. Significant positive correlations between tolerance of different monoterpenes were found, implying the presence of tolerance mechanisms common to a range of compounds.     

Enhancement of attraction and trap catches of the old-house borer, Hylotrupes bajulus (Coleoptera: Cerambycidae), by combination of male sex pheromone and monoterpenes

Monoterpenoid compounds extracted from wood of Scots pine, Pinus sylvestris L, synergized the attraction of the old-house borer, Hylotrupes bajulus (L), to the male pheromone (3R)-3-hydroxy-2-hexanone ((3R)-ketol) + 1-butanol. Glasshouse experiments using ground traps baited with extracts derived from Scots pine wood or the monoterpenes (+)-alpha-pinene, (-)-verbenone, (-)-trans-pinocarveol and (+)-terpinen-4-ol attracted significantly more H bajulus females, but caught fewer of them, than the synthetic pheromone mixture alone. Traps baited with higher concentrations of the monoterpene mixture attracted fewer females than those baited with lower dosages, whilst very high concentrations of the mixture (6-10 vials) caught no insects. However, a combination of (3R)-ketol + 1-butanol or (+/-)-3-ketol + 1-butanol with monoterpenes resulted in the capture of significantly more females than either the sex pheromone or the monoterpene mixture alone. Traps baited with a blend of the male's sex pheromone or the monoterpenes attracted significantly more, but caught fewer, males than females. Here again, a combination of the above blends enhanced the attraction of males significantly when compared with the attractancy of either of the compounds/mixtures used alone.     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

Biological roles of monoterpene volatiles derived from rough lemon (<Emphasis Type="Italic">Citrus jambhiri</Emphasis> Lush) in citrus defense

Volatile compounds of plants, including monoterpenes, are a possible source of signal molecules that induce defense systems to protect plants from tissue damage. Volatile compounds from rough lemon leaves were trapped by solid-phase microextraction fibers in sealed vials, and subsequent gas chromatography–mass spectrometry and gas chromatography analyses identified the profile of the major components, mainly various monoterpenes. Among several monoterpenes examined, citral, citronellal, and linalool significantly inhibited the spore germination and hyphal growth of Alternaria alternata. The effect of linalool was fungistatic, while the effects of citral and citronellal were partially fungicidal. Wounding of rough lemon leaves induced a significant increase in release of monoterpenes. The release of linalool was the most abundant and was 14.5 times that of unwounded rough lemon leaves. Unlike the wounding treatment, microbe attack did not significantly change monoterpene releases, and there was statistically no difference found in the peak areas from microbe-treated and untreated leaves. Linalool, limonene, and β-pinene also had insect-repellant effects on wild-type Drosophila melanogaster. Expression patterns of defense-related genes in rough lemon and rice significantly changed after treatment with vapors of monoterpene volatiles. Taking these results together, monoterpene volatiles are likely to play roles in the defense of rough lemon against microbe and insect pathogens.     

Fireweed (Epilobium angustifolium) as a possible inoculum reservoir for root-rotting Armillaria species

Two glasshouse experiments were conducted to determine the potential for Armillaria species to infect fireweed and subsequently use it as a food base for attacking lodgepole pine. Aspen segments colonized by A. mellea and A. ostoyae were used to inoculate fireweed roots. Both species caused infection, although the former was more pathogenic, attacking 28 of 42 inoculated fireweed roots as opposed to 8 of 60 in the case of the latter species. None of the nine pines inoculated with segments of fireweed roots colonized by A. ostoyae became infected, even though the isolates were pathogenic on pine when aspen segments were used as the food base. In contrast, 8 of 29 pine seedlings inoculated with fireweed colonized by A. mellea were killed, thus suggesting that fireweed could play some role in the epidemiology of this disease.     

Development of a water-soluble preparation of emamectin benzoate and its preventative effect against the wilting of pot-grown pine trees inoculated with the pine wood nematode, Bursaphelenchus xylophilus

Water-soluble preparations have been investigated to develop a trunk injection agent based on the poorly water-soluble anti-nematode emamectin benzoate. Following tests on the phytotoxicity of some solvents and solubilizers and demonstration of the ability of some solubilizers to dissolve emamectin benzoate in water, acetone + methanol was selected as the solvent and Polysorbate 80 as the solubilizer. This water-soluble preparation of emamectin benzoate prevented the wilting of pot-grown 4-year-old trees of the Japanese black pine, Pinus thunbergii, artificially inoculated with the pine wood nematode, Bursaphelenchus xylophilus, at a dose of 20 g emamectin benzoate per cubic metre of pine tree.     

Recovery of Young Olive Trees from <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Natural recovery from wilt disease symptoms was evaluated in young olive trees root dip inoculated with Verticillium dahliae in a growth chamber over a 12 week period and, later on, when the trees were transplanted in a V. dahliae-free soil in a lathhouse during a period of 127 weeks. Recovery in an individual tree was considered when a plant showed symptom remission after having reached a maximum value of symptom severity. Recovery accounted for 53% of 464 trees that showed wilt symptoms during observations in the two environments. The remaining trees died. Recurrent wilt symptoms were not observed in recovered trees, and recovery was usually accompanied by the production of new green tissues. Recovery was clearly higher in trees inoculated with a non-defoliating (ND) isolate (86.4%) of the pathogen than in those inoculated with a defoliating (D) isolate (23.9%). The percentage of recovery and the level of resistance were significantly correlated. Recovery accounted for 92.1% of the cases in resistant and moderately susceptible cultivars, reaching 100% in plants inoculated with the ND isolate (Table 2); meanwhile it was three times lower (30.1% of the plants) in susceptible and extremely susceptible diseased trees. In the lathhouse, periodical tissue isolations for monitoring the progress of infections over a period of 127 weeks in recovered trees, showed that the pathogen could only be isolated from trees 19 weeks after inoculation. Pathogen isolation was significantly higher from susceptible and extremely susceptible cultivars (84.6%) than from resistant and moderately susceptible ones (33.3%). Results showed that if a tree overcomes infection by pathogen from a single inoculation, and it is able to begin a recovery process, it will not express wilt symptoms again in a pathogen-free environment. The pathogen remained inactive or dead over time in recovered trees. Thus, new infections from rootlets would be necessary for new symptom expression. Recovery from Verticillium wilt is an important natural mechanism that occurs in a high percentage of infected olive trees, and can complement the resistance of the cultivar, particularly in conditions of low inoculum densities of low virulence isolates of the pathogen in the soil.     

Further Contributions to the Development of a Differential Set of Pea Cultivars (Pisum sativum) to Investigate the Virulence of Isolates of Aphanomyces euteiches

Common root rot (Aphanomyces euteiches Drechs.) has become a very destructive disease in the French pea crops since 1993. For an accurate investigation of the virulence variability among French A. euteiches populations and between French and foreign populations, a new set of differential pea genotypes was developed. Thirty-three American and European pea lines, displaying different levels of resistance, were screened in a growth chamber against two French isolates. Symptoms (disease severity from 0 to 5, evaluating symptom surface on roots and epicotyl) and percentage of top fresh weight (inoculated/uninoculated top fresh weight ratio) were measured. From this screening 12 relatively resistant lines, from various genetic backgrounds, were identified along with a highly susceptible control. This set of 13 genotypes was inoculated under controlled conditions with 14 isolates from France, Sweden, USA, Canada and New Zealand, to investigate genotype–isolate interactions. Root symptoms were rated (disease severity), and a susceptibility/resistance threshold was established at disease severity = 1. Significant quantitative interactions were observed, and five 'resistance patterns' were identified, leading to a set of six pea genotypes: Baccara (susceptible), Capella, MN313, 902131, 552 and PI180693. Fields trials of this set in 1999 and 2000 gave the same resistance rankings than in growth chamber conditions. This set will allow more accurate assessments of the variability in virulence/aggressiveness of A. euteiches isolates from France and foreign countries, and further investigations of the epidemiological and genetic basis of pea–A. euteiches interactions.     

Aleppo pine provenances vary in susceptibility and secondary chemical response to Gremmeniella abietina infection

Aleppo pine is a thermophilic species that has become a popular tree species in plantings even beyond its natural habitat. In 1999 the pathogenic fungus Gremmeniella abietina was isolated for the first time in Aleppo pine plantations in Spain. The main objective of this study was to analyse the variation in susceptibility to G. abietina (European race, biotype A) infections among trees from five Aleppo pine provenances. Artificial inoculations were performed at two different times in January 2012. The susceptibility of the trees was evaluated by recording disease severity and the length of internal stem necrosis produced by the pathogen. In addition, the concentrations of two flavanone compounds were quantified as putative indicators of resistance. Provenances and G. abietina isolates both significantly affected levels of necrosis although the results were dependent on the time of inoculation. There was a relationship between provenance and resistance, with trees of higher elevation provenances being more susceptible to disease than trees of lower elevation provenances. The amount of naringenin flavanone from trees was significantly different between provenances, suggesting that it is a possible indicator value for resistance. Conversely, no differences were found in the concentration of eriodictyol in trees between provenances. The results show that the provenance‐dependent variation in the susceptibility of Aleppo pine to G. abietina should be considered in selection of seed sources for restoration.     

Dissemination of Pseudomonas savastanoi pv. savastanoi populations and subsequent appearance of olive knot disease

Pseudomonas savastanoi pv. savastanoi (Psv) is the causal agent of olive knot disease. The bacterium survives epiphytically and gains ingress through new wounds where infections and colonization result in knot formation. The natural spread of the bacterium and the subsequent appearance of the disease in olive orchards is poorly understood. The aim of this study was to monitor Psv epiphytic populations in inoculated plants with knots versus non‐inoculated healthy trees within the same orchard over four years. Additionally, disease severity was measured in both inoculated and non‐inoculated control trees. Epiphytic Psv populations moved from inoculated to non‐inoculated trees, although average Psv populations were higher in inoculated trees. Olive knot severity increased over the course of the study in all treatments and cultivars, with all plants reaching a high level of disease by the end of the study. However, the delay in the onset of disease was longer in non‐inoculated than in inoculated trees. Molecular typing of Psv isolates recovered from non‐inoculated control trees confirmed that they were similar to the inoculated strain. These data demonstrate that Psv can move over short distances in olive orchards through dissemination of epiphytic bacteria and suggest a relationship between the presence of epiphytic Psv and the number of knots on trees.     

黑松13个抗病家系对松材线虫的抗性反应及组织病理学观察

 为评估自日本引进的抗性黑松对松材线虫(Pine Wood Nematode,PWN)的抗病性,选用3个不同来源的PWN强毒和中强毒虫株采用皮接法接种,对1.5年生的黑松13个优良家系进行抗病性测定。结果表明,引进的黑松大部分家系对3个PWN虫株都表现出一定的抗病性,但针对不同虫株,抗病各家系间差异明显,其中有7个家系(3、4、5、6、9、11和12号)表现良好。同时,为探讨引进黑松家系感染PWN后的组织病理反应,对12号抗性家系进行组织病理学研究。观察表明,松材线虫在抗性黑松和普通黑松中的纵向扩散速度差别不明显,但其在抗性黑松内的横向扩散速度比在普通黑松内要慢,且抗性黑松体内木射线细胞和围绕树脂道泌脂细胞的薄壁细胞,出现病变要比普通黑松晚;在接种后同一时间取茎段的同一部位发现,抗性松苗组织解剖结构及细胞的病变和破坏程度要比普通松苗轻。     

Effects of defoliation caused by the processionary moth on growth of Crimean pines in western Turkey

An outbreak of the pine processionary moth (PPM), Thaumetopoea wilkinsoni Tams (Lepidoptera: Thaumetopoeidae), began in spring 1998 and lasted 6 years in a Crimean pine (Pinus nigra Arnold) plantation in western Turkey. The effects of PPM on the radial, height and volume growth of Crimean pine trees were investigated by examining the increment losses for three defoliation intensities (groups). PPM activity in Crimean pine stand was assessed through radial increment analysis of cores extracted at breast height. In 2004, increment cores were collected from moderate and high defoliation and low defoliation dominant or co-dominant trees. Based on the sample, annual radial growth indices from 1998 to 2004 were calculated. Growth functions were defined as the cumulative sum of radial, height and volume increment graphically compared between Crimean pine defoliation group sample trees. The sample trees are the same subspecies and varieties. After the defoliations, radial, height and volume growth of low defoliation group trees was found to be significantly greater than that of the other affected groups. During the 1998–2004 period the total radial growth of low, moderately affected and highly affected trees was, respectively, 49, 33 and 31 mm; the total height growth was 3.1, 1.8 and 1.0 m; and the total volume growth was 50, 14 and 10 dm³.