Penetration and initial establishment of Nectria galligena in aspen and peachleaf willow H. ZALASKY Department of Forestry alld Rural Developmel/t of Cal/ada, Forest Research Laboratory, Willl/ipeg, Manitoba Received September 11, 1967 Histological studies of artificially inoculated aspen and peach leaf willow revealed that Nectria galligena penetrated the periderm directly. Penetration and establishment in the petiole base and leaf trace was noticeably more rapid than in the periderm. The resulting lesions in all infections were small and usually latent. Canadian Journal of Botany, 46, 57 (1968) Introduction The methods by which Nectria galligena Bres. enters the host have not been established fully although numerous workers have made important observations on infections in both fruit trees and hardwoods. Goethe's experimental results (1880) with injured and uninjured apple bark of naturally infected twig cuttings maintained in a moist chamber suggested that the germ tubes of conidia and ascospores of Nectria penetrated directly through the epidermis and lenticels. Several studies have indicated that infection by N. galligena does not occur through non-wounded bark; rather it enters through unprotected wounds, small injuries, and leaf scars (Boyce 1961). However, inoculations of intact and wounded bark of aspen, Populus tremuloides Michx., of unspecified age, with mycelium of N. galligena have not been very successful (Lortie 1962-63). The relationship between leaf scars and cankers on young stems of apple was first established by Wiltshire (1921). Later it was found that infections in leaf scars either developed immediately into cankers or were confined by a suberized or gum barrier (Crowdy 1952). In the present study, canker formation caused by N. galligella on aspen was investigated in Saskatchewan and Manitoba. Observations suggested that infection occurs when the trees are young and that bark infections result in latent cankers. Similar observations were made on hardwoods throughout the northeastern Atlantic States (Brandt 1964). In the past, the infection courts at the petiole, leaf trace, and periderm of aspen and peachleaf willow, Salix amygdaloides Anderss., have received little study and their role in the disease cycle remains obscure. The present study is concerned with the initial penetration of the periderm and leaf traces by N. galligena and the early disease symptom. Methods Single ascospore isolates of N. galligena, obtained from perithecia on cankered Populus tremuloides in Manitoba, were maintained on PDA-V/S juice agar. A water suspension of conidia obtained from a pure culture served as inoculum. This was prepared by adding 25 ml of sterilized distilled water to cover the entire surface of a culture in a standard Petri plate. The water was agitated to dislodge the spores and decanted into a beaker. Twelve suckers of 9-month-old aspen and seven of 6-month-old peach leaf willow were obtained from rooted cuttings and maintained in fiats in the greenhouse. The inoculations were made on six dates beginning June 17, 1964, by placing a few milliliters of spore suspension in parafilm funnels (Zalasky 1964) placed on the lower half of the main stem of aspen or on branches of peachleaf willow that had mature periderm. Either the bark alone or the bark and the base of a petiole were immersed. Sterile distilled water was used as a control, and all the funnels were r filled daily for 7 days. The treated plants were maintained up to 11 months after inoculation. Isolations of the fungus from inoculation sites were made 'aseptically' by removing a thin upper layer of infected periderm that had been surface sterilized and rinsed with sterile distilled water. If discolored lesions had formed, the inner tissues of lesions were aseptically transferred to PDA-V/S juice agar. Penetration and initial establishment of the fungus in both hosts were studied using histological methods previously described (Zalasky 1964). The samples were collected periodically, the first sample of willow 15 days, and of aspen 30 days after inoculation. Results Stem infections occurred in 9 out of 13 funnels on aspen, and 6 out of 20 funnels on peachleaf willow. Four suberized cankers, one of periderm and three of leaf trace origin, and numerous
58 CANADIAN JOURNAL OF BOTANY. VOL. 46, 1968 TABLE I Infections caused by Nectria galligena in Populus tremuloides and Salix amygdaloides Funnel inoculations Infections per funnel No. Host Date No. controls Date collected Remarks ---_._- Aspen June 17, 1964 3 3 July 7, 1964 3 3 Oct. 8, 1964 4 4 Nov. 9, 1964 3 3 Peach- Nov. 9, 1964 9 6 leaf Nov. 25, 1964 11 9 willow May 6, 1965 72 periderm lesions Sept. 23, 1964 65 periderm lesions Nov. 24, 1964 Isolations positive Nov. 24, 1964 Isolations failed Dec. 8, 1964 26 periderm lesions Jan. 26, 1965 12 periderm lesions Dec. 8, 1964 12 periderm lesions May 6, 1964 No periderm lesions periderm lesions occurred on aspen; but only one canker of leaf trace origin and several periderm lesions occurred on willow. On November 24, 48 days after inoculation of aspen, attempts were made to culture the fungus from lesions in the periderm and bud tissue at each point of inoculation (Table I); Nectria galligena was not isolated from the lesions. Failure of isolation was probably due to excessive excision of the upper layers containing the mycelial elements. However, on November 24, 140 days after inoculation, N. galligena was isolated from lesions on five stem pieces. All of the controls were free of infection. Penetration Distinct lesions on lateral buds and periderm of aspen and peach leaf willow were first observed 15 days after inoculation. Penetration of the petiole had also occurred 15 days after inoculation. Hyphae were observed radiating peric1inally under the phloem, and aggregations of hyaline hyphae (Fig. 3) were formed intracellularly in the region of the phellogen of both hosts. Suberized Canker Symptom in the Periderm and Leaf Trace In the periderm of both hosts, numerous lesions up to 3 mm in diameter appeared as corky layers (Fig. 1, B) that fissured and turned brown to straw-colored. Within the lesion the suberized corky tissue consisted of isolated portions of dead cortex between successive layers of periderm, the uppermost layers of which fissured parallel to the stem axis. The corky tissues usually were 22 cell layers deep and consisted of two or more periderm layers (Fig. 2). Most of the lesions were superficial and remained small but some of them had coalesced and produced a canker (Fig. 4). The callus fold around the periderm canker was not evident during the 11 months because it is only in an advanced canker that the cambium becomes necrotic and all the tissues collapse. The cankers were few, 6 mm in diameter, and internally necrotic to the centrifugal periphery of the phloem. In the leaf trace a few shallow infections resulted in suberized lesions of the cortex but the bud trace and cambium were unaffected. Several cankers were produced in both hosts in which the lesion at the base of the petiole was dark and necrotic. The leaf detached gradually as the infection progressed into the leaf trace. The small canker was dark and necrotic internally and 7 to 9 mm in diameter. It remained nearly circular as it slowly enlarged. The young canker was slightly sunken and bordered by a raised, partly developed callus fold (Fig. 1, A). Near the surface of the cankers, the fungus produced sporodochia. Histology of Periderm and Leal Trace Infection The germ tubes from spores penetrated the periderm and leaf trace directly and produced hyphae which established themselves inter- and intra-cellularly. In advance of the hyphae in the periderm (Fig. 3) meristematic activity was stimulated to a depth of 50-200 ].l and a series of weak cicatricial zones formed in the inner regions of the cortex. These zones consisted of alternate layers of necrotic or semihyaline cortex, and cork, the cells of which were empty and whose walls were often impregnated with gum. This caused some localized hypertrophy and fissuring. The ramified hyphae in the corky
PLATE I FIG. 1. Cross section of I-year-old artificially infected aspen showing canker development 78 days after inoculation. A, Leaf trace; B, periderm infections. FIG. 2. An enlargement of one of the erumpent spots in Fig. I, B showing A, successive periderm formation and B, fissuring.
ZALASKY: TREE INFECTION BY NECTRIA 59 FIG. 3. Cross-sectional drawing of bark of peach leaf willow showing establishment of hyphae in the periderm, A; dead and usually empty cortex cells, B; and newly formed periderm in the living cortex, C. FIG. 4. Cross-sectional drawing of an aspen stem 335 days after inoculation showing the inner limits of the periderm canker, A; coalesced fissured lesions, B; living cortex, C; phloem, D; and periderm, E. tissue extended through the weak cicatrical zones and inward to the margin of the phloem. In the leaf trace, the hyphae penetrated the cortex at the base of the petiole and invaded all the tissues of the leaf and bud trace, bark, and pith and grew radially, invading the xylem parenchyma, vessels, and xylem fibers 78 days after inoculation. In response to this invasion, most of the parenchyma cells in the bud trace and pith were converted into sc1ereids. A weak cicatrix extended from the cambium and phloem to the periderm at the callus fold bordering the margin of the canker (Fig. I, A). The fungus continued to invade the sapwood and the cambium behind the callus fold and to spread along the cambium in the peripheral area of the canker. Sporodochia occurred in the cankers near the surface at the points of hypha I aggregations. Discussion Microscopic observations of penetration of uninjured periderm and leaf trace give some understanding of the process of infection by N. galligena in aspen and peachleaf willow. The fungus is able to penetrate the semimature and mature periderm of young trees directly. Many infections result in suberized cankers that are slow to develop and few result in active cankers. In suberized cankers the non-infected cortex and phloem hypertrophy and form healing tissue that soon collapses as a result of the necrosis of cells at the inner margin of the lesion. In active cankers necrosis of the cortex and phloem is more rapid and the canker appears sunken and bordered by a callus fold. Several points of discussion are evident. They concern the slow development of cankers, the funnel method of inoculation, and the importance of suberized cankers at the bases of branches. The weak pathogenicity of N. galligena and its apparent inability to invade the periderm and cortex rapidly indicates that the food reserve is low in these tissues. One would expect a higher food reserve in the cortex but the cells of the cortex soon divide in rapid succession to form a weak cicatrix in advance of the fungus. The cells in the affected cortex are empty before they are colonized. However, the fungus is more active once it advances into the phloem and cambium, where the food reserve of nitrogen is high (Kozlowski 1962). The funnel method of inoculation is subject to criticism especially if a section of young stem is kept immersed for several days. The amount of nutrient leaching could appreciably change the
60 CANADIAN JOURNAL OF BOTANY. VOL. 46, 1968 natural resistance of the tissues (Tukey and Tukey 1959). Except for the dry spore method of inoculation, all methods requiring the use of free moisture are subject to the above criticism. Sviridova (1960) has shown that rain water washes large quantities of nitrogen and calcium and a much lower amount of potassium from leaves of European aspen. Potassium, in particular, is washed from young leaves in the spring. Leaching losses from very young leaves are small, but increase with leaf maturity and are greatest when leaves approach senescence. Thus, one could speculate that if leaching of nutrients occurs in the bark, the most leaching would occur in the lower, older parts of the tree. This presumably has some stimulus on growth, sporulation, and succession of fungi on the surface of the bark. The differing rates of leaf scar healing may explain why infected leaf scars either develop immediately into cankers or are confined by suberized lesions. Cankers develop readily in slow-healing leaf scars or in leaf scars with an impaired healing capacity. Lesions in faster healing leaf scars suberize and develop into cankers gradually. This enables some of the buds in the leaf axis to develop and produce small or partly developed branches which die eventually as a result of gradual girdling by a basal canker. Acknowledgment I thank Prof. D. W. French and Dr. P. Manion, University of Minnesota, St. Paul, Minnesota, and Dr. A. Funk, Forest Research Laboratory, Victoria, B.c., for their interest and critical review of the manuscript. BOYCE, J. S. 1961. Forest Pathology. 3rd ed. McGraw Hill Book Co., New York. BRANDT, R. W. 1964. Nectria canker of hardwoods. U.S. Dept. Agr. Forest Pest Leaflet 84, pp. 1-7. CROWDY, S. H. 1952. Observations on apple canker. IV. The infection of leaf scars. Ann. Appl. BioI. 39: 569-580. GOETHE, R. 1880. Weitere Mitteilungen ueber den Krebs der apfelbaume. Landwirt. Jahrb. 2: 837-852. KOZLOWSKI, T. T. 1962. Tree growth. 2nd ed. The Ronald Press Company, New York. LORTIE, E. M. 1962-63. The Nectria canker and its incitant. Dissertation Abstr. 23: 1155-1156. SVIRlDOVA,1. K. 1960. Results of a study of the washing of nitrogen and ash elements from the crowns of trees by rain. Dokl. Akad. Nauk SSSR, 133: 706-708. TUKEY, H. B. and TUKEY, H. B. 1959. Practical implications of nutrient losses from plant foliage by leaching. Am. Soc. Hort. Sci. 74: 671-676. WILTSHIRE, S. P. 1921. Studies on the apple canker fungus. 1. Leaf scar infection. Ann. Appl. BioI. 8: 182-192. ZALASKY, H. 1964. The histopathology of Macrophoma tumefaciens infections in black poplar. Can. J. Botany 42: 385-391.