Influence of Infection Court. Host Vigor. and Culture Filtrates on Canker Production by Botryodiplodia Theobromae Conidia in Sycamore

Some of the factors that influence canker development in American sycamores inoculated with Botryodiplodia theobromae conidia were determined. A combination of B. theobromae culture filtrates and conidia resulted in 100% canker production when Iiitroduced into stem wounds; however. a combination of Cephalosporium diospyri culture filtrates and B. theobromae conidia resulted in no canker production in healthy sycamores. InocU:J.ations with conidia without fungous filtrates resulted in canker development at wounds 0.5 cm below pruned twig terminals but not at wounds on branches with intact terminals. Conidial inocula also induced canker development in Cephalosporium-wilted and water-stressed sycamores. Cankers resulting from conidial inoculations are favored by stress in American sycamores. Plant Dis. Reptr. 62: 934-937. Botryodiplodia theobromae Pat. causes cankers and dieback in sycamores (Platanus occidentalis) growing in plantations (9.10) and natural stands (2.3.12). Many sycamores in east Texas have been killed by a combination of a wilt caused by Cephalosporium diospyri Crandall. and B. theobromae infections (4.5). Botryodiplodia theobromae has a wide host range and geographic distribution (11.13). Its ascogenous stage. Physalospora rhodina (Berk. " Curt.) Cke.. has been reported on ~ spp. and other species (13) but not on sycamore. It is therefore believed that conidia are the major. and perhaps only. spore type for spread in sycamore. Preliminary investigations indicated that B. theobromae conidial inocula could not easily induce canker development in vigorous American sycamores. The objective of this study was to determine some of the required conditions for canker and dieback induction resulting from conidial inoculation of this species. The influence of infection court. culture filtrates. waterstress. and Cephalosporium wilt on canker production by conidia was investigated. MATERIALS AND METHODS Live sycamore twigs (1-2-cm dialn) were cut into 4-6-cm lengths. placed in 125-ml Erlenmeyer nasks. autoclaved for 20 min at 1.05 kg/cm2• and seeded with mycelium from a single-spore culture of a hypervirulent strain of B. theobromae. Mature conidia were produced after 2-3 weeks and were collected by rinsing wiiii sterile distilled water 1-2 hr before inoculation. The hypervirulent strain of B. theobromae used in this study was capable of producing cankers in vigorous sycamores within 4 days after inoculation with mycelium in agar. Fresh and old wounds on 2-year-old sycamores were compared as infection courts for conidia duringthe summerof 1975. Stems were surface-sterilized with 70% ethanol and 1-2-cm incisions were made through the outer xylem with a razor blade. Three incisions (approximately 20 cm apart) were made on each stem. Water-suspended conidia (200-300 per wound) were placed onto fresh and 4-week-old wOlUlds. Controls received sterile distilled water only. Each inoculated wOlUld was covered with sterile water-saturated cotton balls and sealed with clear plastic and masking tape. Initially. 15 inoculations were made for each wound type; two additional inoculation experiments were performed on different trees at I-week intervals to give 45 inoculations for each type of wOlUld. Cell-free culture filtr.ates of.!!. theobromae and.£. diospyri were tested as stimuli for canker production by conidia in sycamore. Filtrates were produced by growing B. theobromae and.£. diospyri separately in 0.8% sycamore wood-chip broth for 15 days on a gyratory shaker (l00 rpm) at 28-300 C. Then broth was separated from mycelium by centrifugation and fUtration through 0.5-,an Millipore filters. Two-year-old pot-planted sycamores were inoculated by Vol. 62. No. H--PLANT DISEASE REPORTER--November 1978 935 making 1-2-cm incisions through the bark and placing 200-300 conidia onto each wound. BO~odiPlodia theobromae filtrates or £. diospyri. filtrates (in cotton balls sat\u'ated with broth were applied over 12 inoculated wounds for 5 days. The ability of conidial inoculations to result in canker development was evaluated on different types of artificial infection courts on 12-yr-old disease-free plantation sycamores in August 1975. Infection courts were: 1) stem wounds made by tangential cuts through outer xylem 0.5 cm below freshly pruned twig terminals. 2) wounds made by cutting through outer xylem of current-year twig growth. 3) wounds made through xylem of second-year twig growth. 4) wounds made by excising leaf buds. and 5) unwounded leaf buds. Twenty infection courts of each type were inoculated with conidia and 20 of each with mycelium in agar. Canker production resulting from conidial inoculations in water-stressed sycamores and a wilting sycamore was investigated also. Pot-planted sycamores were water-stressed by withholding water from them (4-5 days) until incipient wilt symptoms were observed. and then inoculated with conidia. The inoculated sycamores were maintained in a greenhouse at 28-30" C. A branch on a 15-year-old sycamore that had exhibited recurrent Cephalosporium wUt symptoms. but no cankers or dieback. was inoculated in June 1975 by excising its terminal and placing conidia on the wounded surface. Canker and dieback development in the artificially inoculated twig in addition to uninoculated twigs and branches on the same tree were observed closely at I-week intervals for 3 months. RESULTS Cankers were not produced at any of the 90 fresh and 4-week-old wounds on healthy sycamores inoculated with B. theobromae conidia. The fungus was isolated from 93% of the inoculated fresh wounds and 440;. of the inoculated old wounds. however. after 6 months. The fungus was also isolated from 74% of the stem sections 0.5 cm or more above scars left by inoculating fresh wounds and 37% of the stem sections above scars left by inoculation of old wounds. Inoculated sycamores were colonized by the fungus but no cankers developed. Cell-free B. theobromae cultural filtrates applied over 12 wounds inoculated with conidia resulted in 100% canker production after 5 da;ys. but cell-free £. diospyri filtrates applied over 12 inoculated wounds resulted in no canker production. Cankers increased in size even after the B. theobromae filtrates were removed and pycnidia were produced on the cankers after 3 weeks. Necrosis of succulent leaves on sycamore cuttings placed in cell-free B. theobromae filtrates indicated that the fungus probably produced phytoto:xin(s). Necrosis did not occur in leaves on cuttings placed in C. diospyri filtrates or plain broth. Plantation sycamores did not develop cankers where wounded or unwounded leaf buds were inoculated. Botryodiplodia theobromae mycelium in agar induced canker development in wounds on current-year growth. second-year growth. and twigs with pruned terminals. but inoculations by conidia resulted in cankers only on twigs with pruned terminals (Table 1). B. theobromae was isolated from canker-free twigs and wounded leaf buds. however. that were inoculated with conidia. Cankers resulting from inoculations with conidia and mycelium on twigs with pruned Table 1. Canker production by conidia and mycelium of a hypervirulent strain of Botryodiplodia theobromae in different types of artificial infection courts on sycamore twigs. Types of infection courts Woundsc 0.5 cm below pruned twig terminal Wounds on current-year growth Wounds on second-year growth Wounds on leaf buds Unwounded leaf buds Type of Percent inoculuma cankeredb Mycelium 100 Conidia 100 Mycelium 100 Conidia 0 Mycelium 100 Conidia 0 Mycelium 0 Conidia 0 Mycelium 0 Conidia 0 Average canker length (cm)