Haustorial morphology of Cronartium conigenum in naturally infected cones of three Pinus species from Guatemala1

Haustorial morphology of the cone-rust pathogen, C. conigenum, was investigated among naturally infected female strobili of three Guatemalan pines, P. maximinoi, P. pseudostrobus, and P. oocarpa. Among the three pine species, haustorial shapes and sizes were more variable in P. maximinoi and P. oocarpa than in P. pseudostrobus. The haustorial shapes and sizes were more variable in parenchyma cells of the cortex, xylem, and pith than in the cells in the phloem, xylem rays, and tracheids. The haustoria were also present in larger numbers in the parenchyma cells of the cortex, xylem, and pith than in phloem parenchyma cells and tracheids. In living cells, the tips of some haustoria were appressed to the host nuclei.     

Peridermium pini (Pers.) Lev. and the Resin-top Disease of Scots Pine: II. Lesion Anatomy

The stages in the formation of Peridermium pini lesions aredescribed. The hyphae of the parasite were septate, uninucleate,and intercellular, with unbranched haustoria penetrating allphloem cells and xylem parenchyma cells. Lesion extension occurredby apical growth of the hyphae along the outer edge of the functionalphloem. The hyphae stretched unbroken across the active cambium.Active centripetal and longitudinal growth of the mycelium occurredin the resin ducts of the xylem, the hyphae commonly reachingthe pith. Spermogonia were produced in late summer and fall,aecia appeared in spring. In lesion xylem, tracheids were shorter,rays taller, more numerous and wider, but ray cell height remainedunchanged. Fusiform rays increased in number and size and verticalresin ducts increased in number. Changes in xylem tissues didnot become evident until two to four years after invasion ofthe cambium by the mycelium. Invasion of the secondary phloemresulted in an increase in number and size of the longitudinalphloem parenchmya cells, while changes in phloem rays were similarto those in xylem rays.     

Effect of flooding of soil on growth,stem anatomy,and ethylene production of cryptomeria japonica seedlings

Flooding of soil for 55 days altered the rate of growth and stem anatomy of 9‐month‐old Cryptomeria japonica seedlings. Although flooding did not affect height growth it reduced the rate of dry weight increment of seedlings while increasing stem diameter. The reduction in dry weight increment of seedlings resulted largely from decay of roots and, to a lesser extent, from inhibition of growth of roots and needles. The increased diameter growth of flooded seedlings resulted largely from an increase in bark thickness associated with increased phloem production and greater amount of intercellular space. Flooding reduced xylem increment in submerged stems but increased it above the water level because of larger tracheids rather than more tracheids per radial file. Flooding also increased lumen diameters of tracheids, decreased tracheid wall thickness (as a proportion of tracheid diameter), and stimulated formation of axial parenchyma cells in the xylem. Cryptomeria japonica seedlings adapted to flooding by forming adventitious roots, primarily on the original root system and submerged portion of the stem. Such new roots originated in the xylem ray parenchyma. Flooding stimulated ACC synthesis in roots and ethylene production in stems. The role of ethylene in alteration of stem anatomy is discussed.     

Hyphalike haustoria of the haploid stage of Cronartium ribicola in Pinus monticola observed with the scanning electron microscope1

Haustoria of the haploid stage (n stage) of Cronartium ribicola in bark cells of Pinus monticola were studied with the aid of the scanning electron microscope. The morphology of mature haustoria in all kinds of bark cells was consistently filamentous or elongate (hyphalike). Only immature haustoria are short and globose or reniform. Immature and mature haustoria are constricted at the point of passage through the host cell wall. N stage C. ribicola hyphae are characterized as having haustoria that are morphologically similar to intercellular hyphae.     

Xylem dysfunction in Yezo spruce (Picea jezoensis) after inoculation with the blue‐stain fungus Ceratocystis polonica

The blue‐stain fungus Ceratocystis polonica is pathogenic to Norway spruce (Picea abies) in Europe, as well as to Yezo spruce (Picea jezoensis) and Sachalin spruce (Picea glehnii) in Japan. The wilting mechanism in P. jezoensis saplings after inoculation with C. polonica was examined based on anatomical studies of the phloem and xylem of periodically harvested trees. In addition, the course of sap ascent in the trunks was traced by injection of acid fuchsin solution at harvest. As an initial external symptom, needle discolouration was observed. In dye conduction tests, xylem dysfunction in the xylem of inoculated trees became obvious. The dehydrated xylem area (dry zone) had extended more than 20 cm above the inoculation wounds, within 1 month after inoculation. When the sap flow to the branches had nearly stopped, the leaves began to discolour. Hyphae of C. polonica colonized the ray tissue around the inoculation wounds, but were absent at the front of the dry zones. Defence reactions occurred in ray parenchyma cells adjacent to the penetrating hyphae. It is suggested that secondary metabolites, which are formed by the ray cells and epithelial cells of resin canals, are involved in the obstruction of sap flow. Limited necrotic lesions of the phloem and cambium were not associated with foliar symptoms. It is proposed that the dry zone formation caused by C. polonica is the main mechanism leading to tree death.     

Resistance of improved Pinus monticola and some other white pines to the blister rust fungus,Cronartium ribicola,of Hokkaido,Japan

Resistant progenies of Pinus monticola, improved in the northwestern USA, and Asian and European white pines were inoculated with the blister rust fungus, Cronartium ribicola, of Hokkaido. No resistance was observed in P. monticola but the other pines remained healthy. This shows that the resistance of white pines to the blister rust fungus is limited to a particular pathogenic race and a different race of the rust fungus exists in Hokkaido.     

Defence responses in plantation‐grown Eucalyptus globulus and Eucalyptus nitens after artificial fungal inoculation

The formation of reaction and barrier zones was studied in the xylem of Eucalyptus globulus and Eucalyptus nitens tree stems after wounding and artificial inoculation with two white rot fungi. The study had two objectives: to describe host responses in Eucalyptus spp. by light microscopy and to determine whether they would differ in a fungal treatment (wounding and inoculation by one of two fungal isolates) when compared to a control treatment (wounding only). Eucalyptus globulus and E. nitens developed similar reaction and barrier zones. The E. globulus barrier zone was characterized by kino vein formation. In both hosts, the reaction zone was primarily influenced by content and distribution of living tracheids and parenchyma cells within the sapwood. By contrast, the anatomy of the barrier zone showed similarities to the basic xylem structure of each host, except for some cell types that were newly formed (sclereids, kino veins) or increased in number (parenchyma cells, tracheids). Other cell types were reduced in number or completely absent. Host response in terms of barrier zone width appeared to be greater in the fungal than control treatment. Both wood decay fungi appeared to induce a wider barrier zone in both species than that associated with non‐specific damage caused exclusively by wounding. However, the small number of replicates available for this study was possibly insufficient to provide statistical evidence for different barrier zone width between fungal and control treatments.     

Histology of Pinus maximinoi cones infected by Cronartium conigenum1

Apparently healthy and C. conigenum infected female strobili of Pinus maximinoi, collected in Guatemala, were histopathologically studied by light microscopy. Scales from infected cones were fused and fertile scales had aborted seeds. Infected cone scales lacked fibre cells in the cortex, which are abundant in healthy cone scales. Intercellular spaces of cortex contained abundant hyphae compared to phloem and xylem. The cell walls of infected tissues in the ovules were darkly stained, while those of comparable healthy tissue were colourless under Pianeze's IIIB stain. Infected ovules of different developmental stages with nucellus, or nucellus plus spongy tissue, were observed. The cells of each tissue type contained many haustoria and did not develop into female gametophyte and embryo. Ovule colonization by hyphae appeared after pollination and subsequently caused their early abortion.     

Field ready: Development of a rapid LAMP-based colorimetric assay for the causal agent of white pine blister rust,Cronartium ribicola

The invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five-needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non-Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species-specific real-time PCR assay to develop a field-ready loop-mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust.     

Finnish Cronartium ribicola does not infect alternate hosts of Cronartium flaccidum

Susceptibility of the main alternate hosts of Cronartium flaccidum, Vincetoxicum spp. and Melampyrum spp., to C. ribicola was investigated by artificial inoculations on detached leaves in the laboratory and on whole plants in the greenhouse. Neither uredinia nor telia developed on the leaves of either Vincetoxicum hirundinaria, Vincetoxicum nigrum, Melampyrum sylvaticum, Melampyrum pratense, Melampyrum nemorosum, Melampyrum arvense, Melampyrum cristatum or Melampyrum polonicum following inoculation by the 26 aeciospore sources collected from Pinus strobus, Pinus monticola, Pinus flexilis and Pinus peuce in 2000–2003. The results suggest that Finnish C. ribicola does not infect the main alternate hosts of C. flaccidum. Cronartium ribicola and C. flaccidum can thus be identified reliably by inoculations on their main alternate hosts.     

不同产脂量云南松树脂道解剖学比较研究

为确定高产脂云南松的早期选择指标,采用石蜡切片法对不同海拔、不同产脂量云南松的针叶、树干韧皮部和木质部的组成性树脂道特征进行解剖观察,结果表明:高产脂与低产脂云南松的针叶树脂道个数、树干木质部树脂道密度、韧皮部树脂道大小及单位长度树脂道数量存在极显著差异(P＜0.01),但不同海拔之间无显著差异;高、低海拔高产脂云南松针叶树脂道数量分别比低产脂的高38.89％、30.34％,树干木质部树脂道密度分别高58.06％、48.48％,韧皮部树脂道大小分别高32.20％、29.73％,韧皮部单位长度树脂道数量分别高21.95％、38.51％.说明针叶树脂道数量、树干木质部树脂道密度及韧皮部树脂道大小3项指标,也可作为云南松产脂力评价的部分指标.     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

Responses of water-stressedPinus thunbergii to inoculation with avirulent pine wood nematode (Bursaphelenchus xylophilus): Water relations and xylem histology

The effect of water-stress conditioning on water relations and histological features ofPinus thunbergii Parl. inoculated with avirulent isolate ofBursaphelenchus xylophilus (Steiner and Buhrer) Nickle, pine wood nematode, were investigated. Pines were kept under 8 days cycle of severe water stress. One-half of the water-stressed pines died as a result of infection by avirulent pine wood nematode and water stress tended to induce increased susceptibility and/or decreased resistance of pines to avirulent pine wood nematode. In dead pines, the water conducting function of xylem was lost, and all of the parenchyma cells died. In surviving pines, the xylem hydraulic conductivity and the xylem water content were significantly reduced (12 to 23% and 77 to 83%, respectively) compared to controls. Safranin dye perfusion of excised axis stem segments indicated that the water conductance was limited to the very narrow peripheral area of xylem. Embolism caused by cavitation in the tracheids occurred in the central part of xylem and in that dysfunctional region of the xylem the axial parenchyma cells surrounding the epithelial cells, and ray parenchyma cells partly degenerated but the epithelial cells survived. The disruption of tracheid shape observed in surviving pines indicates that avirulent pine wood nematode temporarily disturbed cell division of the cambium. Considering the differences in responses between dead pines and surviving pines after inoculation with avirulent pine wood nematode, the death of water-stressed pines apparently resulted from death of cells, in particular the vascular cambium and the loss of xylem hydraulic function by cavitation.     

Susceptibility and lesion development in Scots pine saplings infected with Peridermium pini in northern Finland

Susceptibility of Pinus sylvestris provenances from northern Finland to Peridermium pini and lesion development caused by the rust were investigated by inoculating 768 saplings. Resin‐top disease symptoms on the stem including stem swelling, occurrence of spermatial fluid and rust aecia, were recorded over a period of 9 years. The disease incidence was low (3.6%), varying within the range of 0–9.4% among pine provenances and 3.4–3.9% among spore sources in 1994–2002. Aecia were produced for the first time 2–4 years after inoculation, appearing mainly in early June, 2–9 years after inoculation. The aecia ruptured in late July to early August, and the overall sporulation lasted for 1–8 years among the pine provenances. Spermatial fluid was observed regularly in the year prior to aecia development 2–7 years after inoculation. Annual fresh swelling was observed for 1–6 years after inoculation. No statistically significant differences occurred neither between the pine provenances, the spore sources nor the years with regard to all the major disease variables. The results suggest that Scots pine provenances from northern Finland are highly resistant to P. pini regardless of the pine provenance or spore source.     

Occurrence of decay-associated xylem suberization in a range of woody species

Suberization responses to naturally-occurring fungal colonization of living xylem tissues were detected in 30 of 37 woody species examined. These responses comprised the suberization of traumatic parenchyma tissues in compartmentalization wall 4 barrier zones laid down in the vicinity of fungally infected wounds, or the suberization of parenchyma cells, tyloses and vessel linings in reaction zone decay margins in fully differentiated sapwood. Additionally, in a few species, suberization of xylem fibres and tracheids was observed where juvenile xylem tissues, apparently still incompletely differentiated, had been exposed by wounding. These xylem suberization responses are considered to increase the decay resistance of the altered cells, and may protect the hydraulic integrity of adjacent tissue, thus contributing to the antimicrobial defence of the living sapwood.     

Susceptibility of Pedicularis spp. to Cronartium ribicola and C. flaccidum in Finland

Aeciospores of Cronartium ribicola and C. flaccidum were collected from several locations in Finland and used to inoculate Pedicularis spp. and some known and suspected alternate hosts in 2008–2009. In all trials, C. ribicola formed uredinia and telia on leaves of Ribes nigrum. No uredinia or telia of C. ribicola formed on older leaves of Pedicularis spp. but both uredinia and telia were found on young leaves of P. palustris ssp. palustris. Cronartium flaccidum produced uredinia and/or telia on leaves of P. palustris ssp. palustris, P. lapponica, Vincetoxicum hirundinaria and Melampyrum sylvaticum. Neither rust infected P. sceptrum‐carolinum, Vaccinium myrtillus, Calluna vulgaris or Ledum palustre. Similar to rusts in Asia and North America, the results showed that European C. ribicola exhibit more variable host reactions and wider alternate host ranges than earlier described. Pedicularis palustris may play a role in the spread of Cronartium in natural forests.     

A study on Xingkai Lake pine gall rust

The Xingkai Lake pine (Pinus takahasii Nakai) gall rust caused byCronartium quercuum(Berk.) Miyabc: Shirai is a serious stem rust in the northeast region of China. The alternate host is oak (Quercus mongolica Fisch). Germination of both acciospores and urediospores was optimal at 12°C and occurred over a range of temperatures, from 4°C to 32°C. Teliospores germinated optimally at 16–18°C and over a range of 8–28°C. All spores germinated best under natural light and dark conditions. Direct light inhibited germination even when followed by darkness. The incubation periods of urediospores and teliospores in inoculation experiments were 6–16 days and 3–33 days respectively. Anatomical studies ofCronartium quercuum galls showed that rust hyphae freely ramify through the intercellular spaces within parenchymatous tissues of the cortex, phloem, cambium and xylem. Hyphae are perennial; large haustoria arc cylindrical with rounded or blunt ends. Among the 12 fungicides tested for control of the rust, pine-tar, pine-tar: diesel oil (1:1, 1:3, 1:5), 75% Bravo emulsion and 70% Mancozeb emulsion applied to the galls produced satisfactory results.     

The effect of bark decortication for hiwada production on xylem and phloem formation in Chamaecyparis obtusa

Of all plant materials used to cover the roofs of traditional Japanese buildings, Japanese cypress (Chamaecyparis obtusa) bark, hiwada, has the longest service life and has been used from ancient times. However, wood and bark properties after hiwada harvest have not been evaluated in detail. We studied whether decortication for hiwada production in winter affected xylem and phloem formation. Decorticated trees still preserved all inner bark and part of the outer bark, and both decorticated and control trees had similar annual ring structures at all stem heights in the xylem and phloem. In both xylem and inner bark, no significant difference in ring width at any stem height was found between annual rings before and after decortication. Thus, this study revealed that the decortication of bark for hiwada production does not affect the formation of xylem and the inner and outer bark if decortication is carried out by highly skilled workers in winter.     

Factors enabling Epinotia granitalis (Lepidoptera: Tortricidae) overwintered larvae to escape from oleoresin mortality in Cryptomeria japonica trees in comparison with Semanotus japonicus (Coleoptera: Cerambycidae)

The factors involved in the successful escape of overwintered larvae of Epinotia granitalis from host oleoresin in the phloem of Cryptomeria japonica were studied. The mortality, period, and body weight of E. granitalis overwintered larvae while feeding on the phloem of C. japonica were compared with the same parameters for Semanotus japonicus larvae. The seasonal fluctuation in the ability of C. japonica to form traumatic resin canals in the phloem was also assessed. The oleoresin mortality of E. granitalis was low compared with that of S. japonicus. Although both species started feeding on approximately the same date in spring, the feeding period of E. granitalis was only about 24 days, showing that E. granitalis faced the danger of host oleoresin for a shorter period than S. japonicus. The larval weight of E. granitalis was consistently greater than that of S. japonicus through the phloem-feeding period of E. granitalis, and the growth rate was also higher in E. granitalis, suggesting that E. granitalis had a better ability to avoid host oleoresin. These results demonstrated that not only the feeding habits, that is, the tendency of a single larva to feed at several sites, but also the shorter feeding period and larger body size were probably the important factors facilitating escape of E. granitalis from host oleoresin mortality. Furthermore, the ability of C. japonica to form traumatic resin canals increased after the starting date of larval feeding in spring. Because E. granitalis finished feeding before the culmination of the ability of C. japonica to form traumatic resin canals, it was likely that the shorter feeding period was the most important factor in enabling E. granitalis to escape from mortality by host oleoresin.     

Factors affecting Chondrostereum purpureum infection of Salix

Factors affecting Chondrostereum purpureum infection of Salix were studied in the laboratory and field. Basidiospores passively entered all xylem tissues but not cambial, phloem or bark tissues. A small number entered stems up to 70 mm from the wound surface although most were retained within tissues 30 mm from the surface. Basidiospores required free water for germination and the optimum temperature for germtube extension was 26°C. Hyphae ramified extensively throughout xylem tissues eventually plugging vessels. Cell walls and starch granules were penetrated with enzymatic assistance. The depth of C. purpureum penetration was affected by wound diameter and inoculum concentration but not stem age. Wounds of Salix were susceptible to C. purpureum throughout the year and greater depth of penetration was obtained using mycelium as inoculum than basidiospores. The depth of C. purpureum penetration decreased with duration of wound exposure prior to inoculation. A characteristic flora of saprophytic fungi and bacteria inhabited wound tissues.