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1.
Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm3 per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.  相似文献   

2.
选择奥地利境内阿尔卑斯山健康欧洲云杉为对象,研究室内接种蓝变真菌(Ceratocystis polonica)引起的寄主树木韧皮部和木质部解剖学特征的变化,揭示蓝变真菌引起欧洲云杉枯萎的机制.结果表明:接种1周后的4株欧洲云杉的木质部组织内,蓝变区域显著增加,4～6周后蓝变区域不再增加;而在接种无菌琼脂的2株对照欧洲云杉的木质部组织内,没有检测到蓝变区域.采用生物化学分析和组织定位技术,确定接种真菌和无菌琼脂的欧洲云杉木质部区域纤维素酶的分布和活性变化.接种2周后剥皮取样检测,接种真菌的4株欧洲云杉的木质部组织内纤维素酶含量大幅度增加,其等电聚焦电泳显示明显的纤维素酶酶带;而在接种无菌琼脂的欧洲云杉木质部区域,纤维素酶含量分布较少,其等电聚焦电泳显示微弱的纤维素酶酶带.进一步证明蓝变真菌分泌的纤维素酶能利用寄主欧洲云杉木质部的纤维素,蓝变真菌是致死阿尔卑斯山境内欧洲云杉的重要病原菌.  相似文献   

3.
Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.  相似文献   

4.
Japanese oak wilt causes widespread oak mortality in Japan. Possible differences in susceptibility to the causal fungus, Raffaelea quercivora, may be due to vessel arrangements in host trees. To clarify whether constitutive defence mechanisms including vessel arrangements or induced defence mechanisms are the main determinants of host susceptibility, we inoculated the fungus into living seedlings or sterilized stem segments of four Japanese fagaceous species. In seedlings, water conductance was assessed with dye. In both seedlings and stem segments, hyphal growth was examined by fluorescence microscopy. In seedlings, the area of non‐conductive sapwood in stem cross sections and hyphal growth differed significantly among species. In the susceptible species Quercus crispula and Quercus serrata, hyphal growth was significantly and positively correlated with the proportion of non‐conductive sapwood. In stem segments, hyphal growth was not significantly different among species or between vessel arrangements and was similar to or greater than that in seedlings. These results suggest that the extent of sapwood colonization by R. quercivora could be used as a marker for susceptibility and that susceptibility is determined mainly by induced defence responses.  相似文献   

5.
In Brazil, the selection and planting of eucalyptus clones resistant to Ceratocystis fimbriata is the pre‐eminent control strategy for the wilt pathogen. However, little is known about host defence responses associated with resistance of eucalyptus to C. fimbriata infection. In this work, enzymatic activity, sugars, lignin, total phenols and phenolic compounds involved in the defence response of eucalyptus clones resistant (RC) and susceptible (SC) to the Ceratocystis wilt were evaluated. Changes were detected in the sugars produced by RC clones, with higher concentrations occurring compared with SC. A similar response occurred with lignin content at 28 days after inoculation (dai) in RC plants. SC plants had an increase in polyphenol oxidase and peroxidase activities at 7 dai. In contrast, RC plants demonstrated high activity of the same enzymes at 2 and 4 dai, decreasing afterwards. There was no difference in phenylalanine ammonia‐lyase activity between resistant and susceptible clones. Hydroxycinnamic acid concentration was higher in RC than in SC; however, there was no difference between RC and SC in flavonoid concentrations. RC had high concentrations catechin, chlorogenic acid and caffeic acid. Histochemical tests demonstrated the presence of phenolic compounds and lignin, at higher intensities, in xylem of RC plants. Inoculation with C. fimbriata induced defence responses in both resistant and susceptible eucalyptus clones, but both the intensity and speed of the responses were higher in RC. Alterations in the concentrations of sugars and lignin, as well as certain enzymes and phenolic compounds, could be used to predict the relative susceptibilities to Ceratocystis wilt of different phenotypes of eucalyptus clones.  相似文献   

6.
Mass mortality of oak trees has been occurring in Japan since the late 1980s. The fungus Raffaelea quercivora has been frequently isolated from discoloured sapwood in dead or wilting trees and inoculation experiments have shown it to be capable of causing wilting and xylem discoloration in several oak species, notably Quercus crispula and Q. serrata. In this study, we inoculated seedlings of six Fagaceae species with R. quercivora and, after 56 days, measured the vertical length of the discoloration and the areas of discoloured and non‐conducting sapwood on stem cross‐sections. The sapwood discoloration and the water non‐conduction areas were larger in Q. crispula and Q. serrata than in the other species.  相似文献   

7.
Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.  相似文献   

8.
To confirm the pathogenicity of a blue stain fungus,Ceratocystis piceae (Münch) Bakshi to the Japanese red pine (Pinus densiflora Sieb. et Zucc.), the responses of healthy young pine trees and stressed trees which were girdled by the half-circumferential girdling technique were investigated by the fungal inoculation test. Although neither of the pine trees inoculated withC. piceae in the non-girdled treatment nor the controls died, mortality of the trees girdled and inoculated withC. piceae was 28.6%. In the pine trees inoculated withC. piceae, the mean area of the necrotic lesion of the sapwood was significantly larger than that of the controls, and the mean of the water pressure potential of the xylem decreased, regardless of the girdling treatment. TheC. piceae was reisolated from the wood pieces near the inoculation points on the inoculated trees, but not from the controls. These results suggest that under strongly stressed conditions, the Japanese red pine trees might have been killed by heavy infestations ofC. piceae carried by bark beetles. A part of this paper was presented at the 103rd Annual Meeting of the Japanese Forestry Society (1992).  相似文献   

9.
Laurel wilt, caused by the fungus Raffaelea lauricola and transmitted by the exotic ambrosia beetle Xyleborus glabratus, has killed members of the Lauraceae plant family throughout the southeast United States. A series of experiments were conducted to examine the effects of inoculum concentration on the development of laurel wilt in swamp bay, Persea palustris, and avocado, Persea americana. In each experiment, host plants were inoculated with aqueous suspensions of 102, 103, 104 or 105 conidia of R. lauricola, and plants were rated periodically for external symptom development (wilting and foliar dieback). At the end of experiments, plants were rated for internal symptoms of the disease (discoloration of sapwood) and assayed for R. lauricola on a semi‐selective medium. Symptom severity in swamp bay was significantly lower for the 102 treatment than at higher (103–105) concentrations, whereas 102 and 103 conidia caused less disease than 104 and 105 conidia in avocado. At the lowest inoculum concentration, 67% of the swamp bay plants and 20% of the avocados died by the time the respective experiments were terminated. The pathogen was recovered from a high proportion of the symptomatic sapwood of swamp bay (100%) and avocado (94%), and sapwood discoloration and recovery of R. lauricola from inoculated stems of swamp bay were highly correlated with recovery of the pathogen and symptom development in roots. Clearly, swamp bay and avocado are very sensitive to R. lauricola. The ability of only 100 conidia of this pathogen to kill these hosts suggests that few individuals of X. glabratus or other ambrosia beetles that carry low levels of the pathogen would be sufficient to transmit conidia that infect and lead to disease development. The results are also relevant to the development of disease‐tolerant host selections, as they indicate levels of the pathogen appropriate for use in screening plants for disease resistance.  相似文献   

10.
Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.  相似文献   

11.
The aim was to determine the inoculation density above which Scots pine (Pinus sylvestris) is overcome by the blue‐stain fungus Ophiostoma ips that is associated with the bark beetle Ips sexdentatus. In north‐western Spain, stems of 16 Scots pines were inoculated at various densities (0, 400, 800 or 1600 inoculi/m2) along circumferential 100 or 150 cm wide inoculation belts. Each inoculum consisted of a 5 mm diameter cylinder of malt extract agar colonized by the fungus. Three months later, all trees were harvested and trunk resinosis and foliage colour were visually assessed. The percentage of healthy, desiccated, resin soaked, and blue‐stained sapwood, as well as growth productivity indices, were calculated from stem disks cut from within the inoculated zone of each tree. Sapwood‐specific hydraulic conductivity (Ks) of each tree was measured in the middle of the inoculated zone. All parameters of tree vigour changed dramatically to the worse when inoculation densities were above 400 inoculi/m2, and foliage changed from green to yellow‐green or yellow when an inoculation density of 800 instead of 400 was used. The percentage loss of sapwood‐specific conductivity (PLC) increased from 30 to 90% and the percentage of healthy, conductive sapwood dropped from 85 to 35% at 800 inoculi/m2. No effect of the width of the inoculation belt was observed, and there was no relationship between tree productivity indices and the level of resistance. A non‐linear negative relationship was found between PLC and the percentage of healthy sapwood. It is concluded that tree resistance was overcome and that trees were going to die when the inoculation density was ≥800 inoculi/m2.  相似文献   

12.
Xylem dysfunction progresses rapidly in Pinus thunbergii infected with pine wilt disease. The present report deals with the timing and process of the extensive dehydration of tracheids by embolism and the subsequent desiccation of the xylem with disease development. An ultrasonic acoustic emission (AE) technique was used to detect embolisms in the xylem of pine trunks. In most of the P. thunbergii saplings inoculated with the pathogen Bursaphelenchus xylophilus, the AE frequency suddenly increased in the second week after inoculation. The high-frequency AE continued for about 3 days and into the nights. Harvesting of specimens at this time revealed that white air-filled patches, representing the dehydrated and dysfunctional areas, had just emerged in the sapwood. The AE events in the night must be due to something other than embolisms in healthy trees. Frequent embolism of tracheids, which was suggested by the elevation of the AE frequency, might occur due to the decrease in the tensile strength of xylem sap. This hypothesis is supported by previously reported data. Host cells that had reacted to infection with B. xylophilus produce and release chemicals which can lower the surface tension of xylem sap. During the second increase of AEs, most of which occurred in the third week, xylem desiccation and needle yellowing progressed. Needle fading then became distinct, and the tree was close to death when the AE frequency dropped during the fourth week. By monitoring the AE, the first physiological abnormality that took place very early after infection was detected.  相似文献   

13.
During spring and summer of 2016, dieback symptoms including blights of leaves, twigs, and acorns were observed on current year shoots of Quercus infectoria in the Zagros oak forests of Iran. The fungus isolated from damaged tissues was identified as Discula quercina based on both morphological characteristics and ITS sequencing. To fulfil the Koch's postulates, a representative isolate was inoculated onto shoots of Q. infectoria and Quercus libani in both laboratory and forest conditions. Responses of the two oak species to inoculation with D. quercina were examined under laboratory and forest conditions in a completely randomized experiment. Discula quercina was clearly involved in oak dieback, and Q. infectoria was more susceptible than Q. libani to damage by the pathogen. This is the first record of the occurrence and pathogenicity of the fungus Discula quercina on Quercus infectoria. The fungus is considered as an emerging pathogen on oak trees in Zagros forests in Iran. Furthermore, the pathogenicity of the Discula quercina on Q. libani under laboratory and forest conditions increases the potential importance of this pathogen in Zagros forests.  相似文献   

14.
Dutch elm disease (DED) spread across Europe and North America in the 20th century killing most natural elm populations. Today, breeding programmes aim at identifying, propagating and studying elm clones resistant to DED. Here, we have compared the physiology and biochemistry of six genotypes of Ulmus minor of variable DED resistance. Leaf gas exchange, water potential, stem hydraulic conductivity and biochemical status were studied in 5‐year‐old trees of AB‐AM2.4, M‐DV2.3, M‐DV2 × M‐CC1.5 and M‐DV1 and 6‐year‐old trees of VA‐AP38 and BU‐FL7 before and after inoculation with Ophiostoma novo‐ulmi. Leaf water potential and net photosynthesis rates declined, while the percentage loss of hydraulic conductivity (PLC) increased after the inoculation in susceptible trees. By the 21st day, leaf predawn and midday water potential, stomatal conductance to water vapour and net photosynthesis rates were lower, and PLC was higher in trees of susceptible (S) genotypes inoculated with the pathogen than in control trees inoculated with water, whereas no significant treatment effect was observed on these variables in the resistant (R) genotypes. Fourier transform infrared spectroscopy analyses revealed a different biochemical profile for branches of R and S clones. R clones showed higher absorption peaks that could be assigned to phenolic compounds, saturated hydrocarbons, cellulose and hemicellulose than S clones. The differences were more marked at the end of the experiment than at the beginning, suggesting that R and S clones responded differently to the inevitable wounding from inoculation and repeated sampling over the experimental course. We hypothesize that a weak activation of the defence system in response to experimental wounding can contribute to the susceptibility of some genotypes to O. novo‐ulmi. In turn, the decline in shoot hydraulic conductivity and leaf carbon uptake caused by the infection further exacerbates tree susceptibility to the fungus.  相似文献   

15.
Cytoskeletal dynamics play a crucial role in pathogen recognition and cell defence during the initial interactions between an invader and plant host. The aim of the work reported here was to characterize how Heterobasidion annosum s.s., Heterobasidion parviporum, and Heterobasidion abietinum affect the microtubules and microfilaments of Pinus sylvestris root cells 12‐, 24‐, 48‐, and 96‐h post‐inoculation. Inoculation of P. sylvestris with H. parviporum or H. abietinum, which have a lower specificity for P. sylvestris than H. annosum s.s, resulted in greater reorganization of host microtubules during the early stages of interaction than inoculation with the more specific H. annosum s.s. In some infected cells, spots of actin aggregates were observed. Disruption of cytoskeletal components by the application of specific cytoskeletal inhibitors facilitated the entry of the H. parviporum and H. abietinum into roots. These results suggest that the P. sylvestris cytoskeleton plays a role in the host response in the initial stages of the host–pathogen interaction.  相似文献   

16.
To reveal the relationship between the susceptibility of Fagaceae species to Raffaelea quercivora Kubono et Shin‐Ito and the tangential expansion of regions of discoloured and non‐conductive sapwood among the species, we inoculated branches of three Quercus species and one Castanopsis species with the fungus. The sapwood around the inoculation hole in all four species became non‐conductive in response to the infection before the discolouration. The expansion of the region of non‐conductive sapwood ceased within 2 weeks after the inoculation. The region of non‐conductive sapwood in Q. crispula and Q. serrata was larger than that in Q. glauca and C. cuspidata var. sieboldii. These results suggest that the region of non‐conductive sapwood expanded soon after the infection by R. quercivora. It was also clear that the transverse non‐conductive sapwood has close relationship with the susceptibility among Fagaceae species to R. quercivora.  相似文献   

17.
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18.
White pine blister rust (Cronartium ribicola) causes extensive damage to white pines and their associated ecosystems across North America. The anatomical and cellular characteristics of C. ribicola colonization in Pinus monticola branch and stem tissues were studied as a basis for understanding host tree reactions that may be related to resistance. Samples examined showed typical fusiform swelling and some had produced aecia. The reaction of phloem and xylem tissues was compared with non-infected tissue using light and electron microscopy. Cortical parenchyma and phloem polyphenolic parenchyma cells underwent mitotic division, cell swelling, and ca sixfold greater accumulation of phenolic compounds in colonized vs. control stems. In the cortex and secondary phloem, haustoria were common in parenchymatous cells, and hyphae were abundant in the intercellular spaces, but cell death was rare, unless aecia had ruptured the stem cortex. Hyphae were also common in xylem rays, tracheids and between tracheids. Disease-induced changes in the cambial zone included development of cambium-derived xylem traumatic resin ducts. Results demonstrate that diverse host defence responses were activated in the bark of apparently susceptible trees, but lack of mechanical damage by C. ribicola to the phenol-containing host cells and the resin duct system allowed extensive colonization and development of aecia despite elicitation of these stem defences. Interactions between P. monticola and C. ribicola are discussed and compared with other conifer–fungus pathosystems.  相似文献   

19.
Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.  相似文献   

20.
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