Etiology of the resinous stem canker ofChamaecyparis obtusa: Cistella japonica as the causal agent

In order to confirm the pathogenicity ofCistella japonica, inoculation experiments were conducted onto several coniferous trees. Resinous lesions similar to those of the resinous stem canker developed by the inoculation withCi. japonica only onChamaecyparis obtusa. Chamaecyparis trees were heavily affected when inoculated with the fungus in November, but slightly in May and August. Resin flows occurred when inocula ofCi. japonica were put into the holes made on stem barks and on pruning scars. Isolates ofCi. japonica varied greatly in the pathogenicity. On the lesions caused by the fungus, resinous and necrotic areas expanded in fusiform or oblong shape, and resin cysts were formed in inner barks. Beneath some necrotic inner barks, sapwood was suppressed in growth for the damages in cambial cells and stained.Ci. japonica was reisolated from the inoculated lesion tissues, though isolation frequency of the fungus became lower in some lesions two or more years after the inoculation. The results of these inoculations proved thatCi. japonica was the causal agent of the resinous stem canker ofCh. obtusa.     

Symptom development of the resinous stem canker caused by inoculation withCistella japonica ontoChamaecyparis obtusa

Cistella japonica was inoculated onto the stems of youngChamaecyparis obtusa trees, and the development of external and internal symptoms was investigated for five years. Most lesions started exuding resin from May to July during the first growing season after inoculation, while a few lesions started exuding resin during the second growing season. Resin exudation lasted for two successive years on most lesions, and during successive three, four, or five years on some lesions. Resin exuded excessively in the secondary phloem of the lesions and resin cysts frequently developed there. The lesions where cambial tissue was necrosed occupied 18% of all the lesions and the resinous areas expanded to a larger size than those where resin simply exuded in the phloem.Ci. japonica was reisolated from some inoculated lesion tissues at a high frequency. The isolation frequency of the fungus from some lesion tissues two and more years later was zero or low and that ofCryptosporiopsis abietina was high. This work was supported by a grant from the Forest Agency of the Japanese Government.     

Methods for inoculum production and inoculation of Cistella japonica,the causal agent of resinous stem canker in Chamaecyparis obtusa

The ascomycete Cistella japonica was cultured on potato dextrose agar medium (PDA) for inoculation into Chamaecyparis obtusa, enabling the development of an inoculation method suitable for use in a breeding programme aimed at selecting for families of Ch. obtusa resistant to resinous stem canker. Using PDA to generate the inoculum resolved the problems encountered with the previously used mixed medium of rice bran and wheat bran, including unfavourable characteristics, uncertain growth of Ci. japonica mycelia, and a complex culturing operation. The inoculation test induced resin exudation similar to that observed in natural infections, and reproduced clonal differences with regard to damage severity.     

Traumatic resin-canal formation caused by inoculation withCistella japonica in secondary phloem ofChamaecyparis obtusa

In the resinous lesion development caused by the inoculation withCistella japonica onChamaecyparis obtusa stem, numerous traumatic resin-canals were formed at certain radial intervals in the secondary phloem. These formation started in the ring grown in the current or the previous year of inoculation and the resin-canals were subsequently formed in the newly grown rings. The resin-canals were arranged in tangential series in one to two lines in an annual-ring. Artificially induced resin-canal formation was similar to that in naturally infected resinous stem canker. The results from the present study reconfirmed thatCi. japonica is the causal agent of the disease.     

Comparison of terpenes in extracts from the resin and the bark of the resinous stem canker of Chamaecyparis obtusa and Thujopsis dolabrata var. hondae

A monoterpene and 15 diterpenes were isolated from the ethyl acetate extracts of the bark-glued resin from the resinous stem canker ofThujopsis dolabrata var.hondae Makino. A monoterpene (nezukone20) and 4 diterpenes (acetyl torulosol5, acetyl isocupressic acid8, acetyl abietinol11, and 7-methoxytotarol18) were characteristic constituents of the ethyl acetate extracts but were absent in then-hexane extracts from the resinous stem canker ofT. dolabrata var.hondae. These terpenes were first isolated fromT. dolabrata var.hondae andT. dolabrata. The available literature suggests that diterpene18 is a new compound. The resinous stem canker ofChamaecyparis obtusa Endlicher contained larger amounts of manool1,trans-communic acid6, and ferruginol12 and smaller amounts of isocupressic acid7 and abietinol10 than the resinous stem canker ofT. dolabrata var.hondae. The concentration of18 was less than 2% in the extracts, and the resinous stem canker ofC. obtusa lacked this compound. The resinous stem cankers ofC. obtusa andT. dolabrata var.hondae provided extracts 15.6 and 4.96 times, respectively, heavier than the healthy ones. Large differences in the ratios and compositions of terpenes were also observed between the resinous stem canker and the healthy trees. Terpenes isolated from the extracts contained many kinds of diterpene, especially the labdane-type diterpenes, in these diseased trees. These results suggest that the presence of labdane-type diterpenes is closely associated with the resinous stem canker or the causal fungi of this disease.Part of this report was presented at the 50th annual meeting of the Japan Wood Research Society, Kyoto, April 2000     

Relationship between canker size and wood starch in American chestnut

Chestnut trees (Castanca dentata) were wounded in May and inoculated with Cryphonectria parasitica. In August, canker area was measured and the surfaces of longitudinal dissections of stem wood were stained with I₂KI to reveal the presence of starch. Sections with the most starch had smaller cankers than sections with no starch. Results show that starch reserves were related to canker size and therefore may play a role in the tree's ability to respond to infection.     

Distinction of resin compounds between the healthy bark and the resinous stem canker of <Emphasis Type="Italic">Thujopsis dolabrata</Emphasis> var. <Emphasis Type="Italic">hondae</Emphasis>

Methyl esters of higher fatty acids were detected from the healthy bark of Thujopsis dolabrata var. hondae but not from the bark of the resinous stem canker of T. dolabrata var. hondae. This difference enabled us to distinguish healthy trees from diseased ones. Fourteen diterpenes were also isolated from the n-hexane extracts of the bark-glued resin taken from the resinous stem canker of T. dolabrata var. hondae. Of these diterpenes, abietane diterpenes [abieta-7,13-diene (1), abietinol (2), dehydroabietinol (4)], pimarane diterpene [sandaracopimaric acid (8)], and labdane diterpenes [manool (10), torulosol (11), torulosal (12), cupressic acid (13)] were first isolated from T. dolabrata var. hondae.Part of this paper was presented at the 47th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Lesion development in stems of rough‐ and smooth‐barked Eucalyptus nitens following artificial inoculations with canker fungi

A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16‐year‐old plantation trees. In a first trial cambium bark wounds on smooth‐ and rough‐barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm² 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm²). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to post‐penetration hyphal spread in comparison with rough bark. It is suggested that at a pre‐penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.     

Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

Growth and physiological responses of Quercus brantii seedlings inoculated with Biscogniauxia mediterranea and Obolarina persica under drought stress

Charcoal canker of oak is a common disease in the western regions of Iran, which has widely spread due to climate change and increasing drought in recent years. Biscogniauxia mediterranea and Obolarina persica are known as two agents of oak charcoal canker in Zagros forests causing the dieback of oak trees. The effects of charcoal disease agent pathogens on the growth and physiological response of two‐year‐old seedlings of Quercus brantii were evaluated under drought stress in greenhouse over a period of nine months. Survival was 21.7% lower in seedlings inoculated with B. mediterranea subjected to drought stress compared with control treatment. The length of lesions on stems was 68% more under charcoal pathogens in drought stressed compared with the length of lesions caused by charcoal pathogens only. On the other hand, stem lesion length caused by B. mediterranea was 25% greater than those caused by O. persica. Some morphophysiological characteristics were affected by charcoal pathogens alone and/or in combination with drought. Diameter, height and biomass decreased the most in seedlings inoculated with charcoal pathogens under drought stress. Pathogenic agents alone and in combination with drought stress reduced photosynthetic rates, stomatal conductance, transpiration, the maximum Rubisco activity (V_cmax), maximum photochemical efficiency of photosystem II (F_v/F_m) and xylem water potential. In general, drought increased the aggressiveness of fungus and intensification of their destructive effects in Quercus brantii. Moreover, there was no significant difference between the effect of B. mediterranea and O. persica on the morphophysiological parameters studied.     

First report of Teratosphaeria zuluensis causing stem canker of Eucalyptus grandis in Uganda

Teratosphaeria stem canker is one of the most important diseases to have emerged on non‐native plantation‐grown Eucalyptus trees globally. In 2012, Eucalyptus grandis trees with typical Teratosphaeria stem canker symptoms were observed in Uganda. Multigene sequence analyses of isolates from these cankers led to the identification of T. gauchensis, previously recorded in Uganda, and T. zuluensis. This study represents the first report of T. zuluensis in Uganda. Furthermore, this is the first report of the co‐occurrence of T. zuluensis and T. gauchensis in a single area.     

Effectiveness of a detached‐leaf assay as a proxy for stem inoculations in backcrossed chestnut (Castanea) blight resistance breeding populations

A recently developed detached‐leaf blight resistance assay has generated interest because it could reduce the amount of time needed to evaluate backcrossed hybrid trees in the American chestnut blight resistance breeding programme. We evaluated the leaf inoculation technique on a sample of advanced progeny from the Indiana state chapter American Chestnut Foundation breeding programme, along with susceptible American chestnut (Castanea dentata), the recurrent parent, and resistant Chinese chestnut (Castanea mollissima), the donor parent for blight resistance. In experiments over 2 years using two pathogen isolates, we found no biologically meaningful relationship between leaf lesion size and the size (length and width) or severity (1–5 canker severity rating) of stem cankers on 5‐year‐old trees. Chinese chestnuts did develop significantly smaller leaf lesions than American or backcrossed chestnuts. We conclude that while the detached‐leaf assay may have utility in some chestnut breeding applications, it is not a suitable proxy for the established practice of stem inoculations.     

Evidence for the occurrence of induced resistance to pitch canker,caused by Gibberella circinata (anamorph Fusarium circinatum), in populations of Pinus radiata

Pitch canker, caused by Gibberella circinata, was discovered in California in 1986. Although initially quite damaging to Monterey pines (Pinus radiata), the severity of pitch canker has moderated in areas where the disease was first observed and some trees appear to have recovered completely. The absence of symptoms on trees that were once severely affected implies they have become more resistant to the disease. Experimental work has shown that P. radiata can manifest systemic induced resistance (SIR) in response to infection by the pitch canker pathogen and observations of disease remission may indicate that SIR is operative under natural conditions as well. As a test of this hypothesis, the susceptibility of trees in remission was assessed by inoculating them with G. circinata and recording the extent of lesion development. In addition, randomly selected trees in areas that differed in residence time of pitch canker were inoculated to determine if trees with a longer period of exposure to the pathogen were more resistant to the disease. The results of these tests showed that 89% of trees observed to be in remission sustained very limited lesion development, consistent with resistance to pitch canker. Furthermore, trees in areas where pitch canker was well established tended to be more resistant than trees in areas where the disease was of more recent occurrence. In sum, these findings support the view that SIR occurs in P. radiata and is contributing to a moderation of the impact of pitch canker under natural conditions.     

Traumatic resin canals as markers of infection events in Douglas‐fir roots infected with Armillaria root disease

In response to an infection, traumatic resin canals (TRCs) are formed in the roots of many conifers, which may be used to determine the timing and sequence of infection events essential for epidemiological studies of root diseases. Juvenile Douglas‐fir (Pseudotsuga menziesii) tree roots at coastal and interior sites in British Columbia were wounded at various times of the year or were inoculated with an isolate of Armillaria ostoyae, and root sections were taken to determine the timing and extent of TRC formation. Naturally infected Douglas‐fir were also examined to determine the extent of the TRCs in infected and uninfected roots on infected trees and in the lower stem. Wounds made in March and October had poor or no TRC formation while the summer wounds responded strongly and were associated with resin soaking. Roots wounded in October did not respond until the following year in all trees except one. Trees produced TRCs and resin soaked tracheids at all times in response to the fungal inoculations. The most striking difference between wounding or fungal inoculation was the multiple bands of TRCs produced in response to the fungus. TRCs at natural A. ostoyae infections were found 92% of time in roots at the stem junction and 74% of the time in the stem at soil line. TRCs were produced in uninfected roots on infected trees but disappeared with increasing distance from the initiating lesion. TRCs can be used to time yearly and seasonal root infections when they can be traced from an identified lesion.     

Host responses to natural infection by Cytonaema sp. in the aerial bark of Eucalyptus globulus

The chemical and anatomical host responses to natural fungal infection by Cytonaema sp. in the aerial bark of 3‐year‐old Eucalyptus globulus plantation trees were examined. The lesion margin (LM) of the canker‐infected bark was characterized by the formation of a layer of dark extractives visible to the naked eye. Chemical analysis of the LM by gas chromatography‐mass spectrometry (GC‐MS), high‐performance liquid chromatography (HPLC) with ultraviolet detection and HPLC‐MS using negative ion electrospray ionisation indicated the presence of a range of compounds including hydrolysable tannins, polymeric proanthocyanidins, flavonoid glycosides, formlyated phloroglucinol compounds and volatile terpenes. These compounds were either undetectable in healthy tissue or present at significantly lower concentrations than in the LM. The LM of the canker‐infected bark was morphologically distinct from healthy phloem, its characteristics varying depending on severity of canker infection. In superficial infections in which only the phloem was affected, the following LMs were observed: (i) a continuous wound periderm of multiple layers, or (ii) an incompletely differentiated and discontinuous wound periderm. In cases of severe canker infections in which the vascular cambium had been killed, the new phloem formed subsequently contained traumatic oil glands in addition to the responses observed for superficial canker infections. All LMs were characterized by the formation of new parenchyma cells that stained positive for the presence of polyphenols. The significance of the chemical and structural responses as defence mechanisms against fungi causing stem canker is discussed.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

Pathogenicity of Ceratocystis resinifera to Norway spruce

The blue‐stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single‐point inoculations on young trees (two inoculations per tree on 14‐year‐old trees) and mass‐inoculations on older trees (～200 inoculations per tree on 34‐year‐old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue‐stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue‐stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non‐pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.     

Characterization of Lasiodiplodia theobromae (Pat.) Griffon & Maubl causing stem canker on Teak (Tectona grandis L.) in Ecuador

Teak is a very important tropical timber in Ecuador. In 2017, teak samples displaying stem canker symptoms were collected in two provinces of Ecuador. From 11 symptomatic trees, 11 isolates resembling a species of Lasiodiplodia were obtained. All isolates obtained induced stem canker on teak plants after artificial inoculation, confirming them as the cause of the observed canker symptoms. Bayesian inference with concatenated sequences of complete ITS and partial TEF-α and β-TUB gene sequences from two representative isolates clustered teak isolates with other sequences of Lasiodiplodia theobromae available in Genbank. This is the first report of L. theobromae causing stem canker on teak plants in Ecuador.