Solid Substrate Production of Alternaria alternata f. sp. sphenocleae ...

Biocontrol Science and Technology (2000) 10, 399 ± 409

Solid Substrate Production of Alternaria alternata f. sp. sphenocleae Conidia RHOMELA F. MASANGKAY, TIMOTHY C. PAULITZ, STEVEN G. HALLETT and ALAN K. WATSON Graduate Student, Associate Professor, Assistant Professor, and Professor, Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, QueÂbec, Canada, H9X 3V9 (Received for publication 25 October 1999; revised manuscript accepted 21 February 2000)

Sphenoclea zeylanica ( gooseweed), a major weed of paddy rice in Southeast Asia, is one of the targets in a biological weed control research program in the Philippines. A fungal pathogen, Alternaria alternata f. sp. sphenocleae, is being evaluated as a biological control agent for this weed. The feasibility of solid substrate fermentation for the mass production of A. alternata f. sp. sphenocleae has been examined. Conidia production and virulence of A. alternata f. sp. sphenocleae were aVected by temperature, light, and incubation period. Abundant conidia were produced under continuous light on seeds of sorghum, hard red spring wheat, and barley at 28ë C. The greatest number of conidia was produced on sorghum seed followed by barley and oats seeds at 28ë C exposed to near-ultraviole t (NUV). More conidia were produced at 28ë C under NUV light on sorghum, barley, oats, and hard red spring wheat seeds, cornmeal, and polished rice grains than on the other substrates. Less conidia were produced on these substrates under light. At 28ë C, large numbers of virulent conidia were produced on sorghum seeds after 4 weeks of incubation under either constant light or dark. A mix of equal quantitie s of sorghum seeds and water (w/v) maximized conidial production . Conidia produced on sorghum seeds had a shelf life of at least 12 months when stored in production Xasks under room conditions (246 2ë C). The use of sorghum seeds as a solid substrate for production of A. alternata f. sp. sphenocleae could be a feasible method to produce conidia in a village co-operative or cottage industry type scenario in Southeast Asia. Keywords: bioherbicide, cottage industry, gooseweed, solid substrates, sporulation INTRODUCTION Gooseweed (Sphenocle a zeylanica Gaertner) is an annual herbaceous broadleaf weed species native to tropical Africa (Holm et al., 1977; Waterhouse, 1993) but distributed in all Correspondence to A. K. Watson. Agronomy, Plant Physiology, and Agroecology Division, International Rice Research Institute, MCPO Box 3127, Makati City 1271, Philippines. Tel: + 63 2 845 0563; Fax: + 63 2 845 0606; E-mail: [email protected] Present address for S. G. Hallett. Tropical and Sub-Tropical Weed Research Unit, School of Land and Food, University of Queensland, Gatton College, Queensland 4345, Australia. ISSN 0958-3157 (print)/ISSN 1360-047 8 (online)/00/040399-11

2000 Taylor & Francis Ltd

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R. F. MASANGKAY ET AL.

subtropical and tropical regions of the world. It is a serious weed of rice (Oryza sativa L.) (Holm et al., 1977) and is among the 32 worst agricultural weeds in Southeast Asia (Waterhouse, 1993). S. zeylanica is one of the nine major weeds of rice included in the biologica l weed control research program that began in 1991 at the International Rice Research Institute (IRRI) and the University of the Philippines, Los BanÄos, Laguna, Philippines, in collaboratio n with McGill University, MontreÂal, QueÂbec, Canada (Watson, 1991; Bayot et al., 1994). In 1991, a fungal pathogen, Alternaria alternata (Fr.) Keissler f. sp. sphenocleae (IMI 360160), was isolated from blighted S. zeylanica collected from a rice ® eld near Los BanÄos. Laborator y and ® eld studies have demonstrated that this pathogen is a promising bioherbicid e candidate (Bayot et al., 1994; Mabbaya d & Watson, 1995; Masangkay et al., 1999a,b) . This isolate was previously shown to sporulate on half-strength potato dextrose agar (12 PDA) and sorghum seeds in either light or dark conditions (Mabbaya d & Watson, 1995). Technologie s to mass-produce and deliver the biocontrol `product’ to the farmers must be developed and optimized. Two laborator y methods were superior for producing large quantities of virulent conidia (Masangkay et al., 2000). These were half-strength PDA at 28ë C under constant nearultraviolet (NUV) light, incubated for 4 weeks and a modi® ed sporulatio n medium (S-medium) technique. These studies helped to de® ne preferred conditions for sporulation, but agar media are time-consuming to prepare, expensive, and certainly not feasible for the large-scale production of conidia for ® eld studies. Fungi can be divided into those that sporulate readily in liquid culture and those that do not (Stowell et al., 1989). Submerged liquid production systems are preferred for mass production of bioherbicide s (Churchill, 1982; Stowell et al., 1989), but Alternaria spp. do not sporulate in liquid culture (Walker, 1982). A biphasic methodology, combining liquid and solid systems involvin g mycelia production in submerged liquid culture, followed by drying macerated harvested mycelia in shallow pans produced profuse sporulatio n of some Alternaria spp., including A. cassiae (Walker, 1980; 1982). Signi® cant eVorts to commercialize A. cassiae (CASST) using this production method failed. Many fungi are very adaptable to solid substrate fermentation (Templeton & Heiny, 1989) and agricultural-base d products have been used as substrates to produce inoculum of several bioherbicid e fungi (Morin et al., 1989; Connick et al., 1990; Zhang & Watson, 1997). This method is very common for the mass production of sporeproducing organisms used to transform organic compounds (Mudgett, 1986) and for microbial insecticides (Soper & Ward, 1981; Feng et al., 1994). The objective of this study was to investigate the production of A. alternata f. sp. sphenocleae on agricultural-base d solid substrates. MATERIALS AND METHODS Pathogen Maintenanc e Monoconidia l isolates of A. alternata f. sp. sphenocleae (IMI 360160) on half-strength (12 ) PDA were imported from IRRI, Philippine s into the quarantine facility of McGill University, MontreÂal, QueÂbec, Canada. These monoconidial isolates were inoculated onto S. zeylanica plants, re-isolated, and maintained as stock cultures on 12 PDA in small vials under mineral oil at 4ë C (Tuite, 1969). Small pieces of mycelium from the stock cultures were aseptically transferred to cooled PDA (20 ml) in plastic Petri dishes (90 mm diameter) which were sealed with Para® lm (American Stantard Can, Greenwich, CT, USA), and incubated at 28ë C under constant NUV light from J-05 lamps (UVP Inc., Circleville, OH, USA) for 5± 7 days. Agar plugs (4 mm diameter) from the margins of young actively growing colonies were used as seed inoculum (Tuite, 1969). EVect of DiVerent Solid Substrates, Temperature and Light Condition s on the Production of Conidia Seeds of barley (Hordeum vulgare L.), black-eyed bean [Vigna unguiculat a (L.) Walp.], chickpea (Cicer arietinum L.), maize (Zea mays L.), and cornmeal, and cracked maize,

MASS PRODUCTION OF ALTERNARIA

401

cowpea [Vigna sinensis (L.) Engl.], millet [Setaria italica (L.) Beauv.], mungbean [Vigna radiata (L.) R. Wilcz.], cracked mungbean, oat (Avena sativa L.), peanut [Arachis hypogaea (L.) R. Wilcz.], polished rice grains (Oryza sativa L.), sorghum [Sorghum bicolor (L.) Moench], leaves, stalks, soybean [Glycine max (L.) Merr.], common sun¯ ower (Helianthus annuus L.), durum wheat (Triticum durum Desf.), and hard spring red wheat (T. aestivum L.) were evaluated as substrates for conidia production of A. alternata f. sp. sphenocleae. All the seeds and cornmeal were obtained from various commercial suppliers; mature sorghum leaves and stalks were collected from plants grown on the Horticulture Farm of McGill University. Leaves were cut into approximatel y 1 cm - 2 pieces and dried at 60ë C for 7 days. Dried leaf material (1 g) was placed into each 250 ml Erlenmeyer ¯ ask and soaked with 10 ml of de-ionized water overnight before autoclaving. Sorghum stalks were cut into 1 cm lengths and dried at 80ë C for 7 days. Dried stalk materials (2 g) were placed into each 250 ml Erlenmeyer ¯ ask and soaked in 10 ml of de-ionized water overnight before autoclaving. For the other substrates, 20 g of each substrate was combined with 20 ml of deionized water in a 250 ml Erlenmeyer ¯ ask for 1 h. Flasks were closed with cotton plugs, autoclaved for 17 min at 120ë C and 100 kPa, and subsequently shaken by hand to disperse the particles. After cooling, an agar plug (4 mm diameter) from 7-day-old PDA cultures was placed in each ¯ ask under aseptic conditions. Incubation temperatures were 24, 28 and 32ë C under constant light (400 l Em - 2 s - 1 PAR). Exposure to constant NUV at 28ë C was included as an additiona l treatment. Inoculated ¯ asks were shaken by hand every second day for 14 days to prevent aggregation of solid particles and to improve aeration (Mudgett, 1986). Conidia were harvested 4 weeks after inoculatio n (WAI) by adding 50 ml of sterile de-ionized water to each ¯ ask, and shaking the ¯ asks on a rotary shaker at 200 rpm for 10 min at room temperature (246 2ë C). The ¯ ask contents were then poured through a 250 mm plastic sieve lined with two layers of sterile cheesecloth to remove the mycelium. Conidial concentration was determined using a haemacytometer and a light microscope ( 3 100 magni® cation). Eight readings for each ¯ ask were recorded and averaged. EVect of Temperature, Light Conditions and Incubation Period on the Number and Virulence of Conidia Produced on Sorghum Seeds Flasks of sorghum seed substrate were prepared and inoculate d with seed culture of A. alternata f. sp. sphenocleae as previously described. Incubation temperatures were 24, 28 and 32ë C and light conditions were constant dark (D), constant light (L), and 12 h of alternating light and dark (L /D) conditions. Conidia were harvested 1, 2, 3 and 4 WAI, and the number and virulence of conidia produced were determined. Conidial production was determined as described above and conidial virulence assessed, only on treatments that produced more than 1 3 10 5 conidia g - 1 substrate, as percentage reduction in dry weight of S. zeylanica 14 days after inoculatio n (DAI) (% reduction in dry weight 5 [(dry weight of check plant 2 dry weight of treated plant) /dry weight of check plant] 3 100). Dry weight was measured after cutting plants at soil level and drying stem and leaf tissues in paper bags for 6± 7 days at 45ë C. Dead leaves and dead portions of stems were not included in the dry-weight measurements. S. zeylanica seeds were soaked with 95% hydrochlori c acid (HCl) for 10 min, washed under continuously running distilled water for 30 min, and soaked in distilled water at room temperature (246 2ë C) for 12 h. Seeds were sown in black plastic potting ¯ ats (25 3 50 3 6 cm) 2/3 ® lled with sterilized soil mixture consisting of three parts garden soil, three parts Pro-mix (Premier Horticulture Inc., Red Hill, PA, USA), two parts vermiculite, and one part sphagnu m peat moss. Soil was ® rmed and watered until saturation. Seeded trays were placed in a controlled environment chamber [Conviron E 15, Winnipeg, MB; 32 / 24ë C day/night, 400 l Em - 2 s - 1 photosynthetically active radiation (PAR), 12 h day - 1 , and 70± 80% relative humidity (RH)] until the plants were ready for transplanting . RH was maintained with a humidi® er, soil was kept saturated, and the soil surface was misted every 12 h using a hand-held atomizer.

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Healthy seedlings were selected 21 days after sowing and transplanted into transparent plastic containers (10.5 cm diameter 3 7.5 cm high) half ® lled with sterilized moistened soil mixture. Three plants were transplante d in each container and fertilized with 10 ml of a solution containing 1.25 g l - 1 of 20-20-20 (N-P-K) fertilizer. Containers were returned to the controlled environment chamber and ¯ ooded with water to a depth of 2± 3 cm throughou t the experimental period. Each pot of plants (19± 20 cm high) was inoculated with 10 ml of an aqueous suspension of 1 3 10 5 conidia ml - 1 and 0.01% Triton X-100 (polyethylene glycol tert-octylphenyl ether) as a wetting agent, using a hand-held atomizer. Control treatments were sprayed with deionized water containing the wetting agent. After spraying, pots were placed in a dark dew chamber (Percival, Scienti® c Inc., Boone, IA, USA) with 100% RH at 24ë C for 8 h and were then transferred to the controlled environment. EVect of Moisture Content and Quantity of Sorghum Seeds on Conidial Production Flasks of sorghum seed substrate were prepared and inoculated as previously described, except that moisture content (10, 20 and 30 ml) and quantity of sorghum seeds (10, 20 and 30 g) were compared. Percentage moisture content (MC) of the seeds after autoclaving was evaluated on a wet basis using the formula: MC (%) 5 (g H 2 O /g wet sorghum seed 3 100). Inoculated ¯ asks were incubated at 28ë C under constant light. Conidia were harvested at 4 WAI and conidial production was evaluated as described above. EVect of Storage Period on the Number, Germination and Virulence of Conidia Produced on Sorghum Seeds Flasks of sorghum seed substrate were prepared and inoculated as described above. Inoculated ¯ asks were incubated at 28ë C under constant light for 7 days and, subsequently, placed on the laborator y bench (246 2ë C) until harvest. Conidia were harvested after 1, 2, 3, 6, 9 and 12 months of storage. Production and virulence of the conidia were determined as described above. Conidial germination was determined by placing 50 l l droplets of conidial suspension (1 3 10 5 conidia ml - 1 in sterile deionized water) on 1.5% water agar disks (20 mm diameter). Droplets were allowed to air dry for 5 min, covered with a cover slip, and incubate d in Petri dishes at 24ë C under constant darkness for 8 h. Each treatment replicate had two sample units (agar disks) for each conidial suspension. Germinating conidia were killed and stained with lactophenol-cotton blue (Tuite, 1969) before counting microscopically ( 3 100 magni® cation). Conidia were considered to have germinated when the length of the germ-tube was greater than the width of the conidium . Several randomly selected ® elds of view were observed per sample until a total of 100 conidia/agar disk had been assessed for germination. Comparison of Production, Germination and Virulence of Conidia on DiVerent Media Two agar media [12 PDA and V-8 juice agar (VJA)] and sorghum seed were used for comparison. Plates and ¯ asks were inoculated with seed inoculum from starter cultures and incubated at 28ë C in the dark. Conidia were harvested at 4 WAI. Production, germination and virulence of the conidia were determined as described above. Data Analyses All experiments were performed twice and laid out in either a randomized complete block design (RCBD) or a completely randomized design (CRD) with three replicates. Data from conidial counts were subjected to log (1 + x) transformation for zero values and log (x) for non-zero values and percentage data were arc sine transformed before analysis (Gomez & Gomez, 1984). Results from the two trials were pooled if homogeneity of variances was con® rmed by Bartlett’s test (Gomez & Gomez, 1984). One-way analysis of variance (ANOVA), two-way ANOVA, and three-way ANOVA were performed depending on the number of factors used. All analyses were conducted using Sigma Stat Statistical Software

403


Version 2.0 (SPSS Inc., Chicago, IL, USA, 1997). Treatment means were separated using Tukey’s test (P < 0.05).

RESULTS EVect of DiVerent Solid Substrates, Temperature and Light Conditions on Conidial Production Twenty solid substrates were evaluated for the mass production of A. alternata f. sp. sphenocleae at diVerent temperatures under constant light. There was a signi® cant (P < 0.001) interaction between these substrates and temperature for conidial production. The best temperature for conidial production was at 28ë C wherein the highest number of conidia (21.0 3 10 4 g - 1 substrate) was produced on sorghum and hard red spring wheat seeds. Under constant light condition, very few conidia (0.1 3 10 4 to 11.2 3 10 4 g - 1 substrate) were produced at 24ë C on any of the diVerent substrates, and no conidia were produced at 32ë C. There were signi® cant diVerences (P < 0.001) between the diVerent substrates when exposed to constant NUV or continuou s light at 28ë C. Under continuous light, the most abundan t conidia were produced on seeds of sorghum, hard red spring wheat, barley, and oats (10.6 3 10 4 to 21.0 3 10 4 g - 1 substrate) and the rest of the substrates gave relatively poor conidial production (0.1 3 10 4 to 5.5 3 10 4 g - 1 substrate) (Table 1). Under NUV light, maximum production of conidia was on sorghum seeds followed by oat seeds and barley seeds. Exposure to NUV produced signi® cantly (P < 0.05) more conidia on seeds of barley, oat, sorghum, and hard red spring wheat, cornmeal, and polished rice grain than constant TABLE 1.

In¯ uence of various solid substrates and light on production of A. alternata f. sp. sphenocleae conidia at 28ë Ca Number of conidia g Lb

Substrate Seeds Sorghum Hard red spring wheat Barley Oat Durum wheat Millet Mungbean Maize Soybean Black-eyed bean Chickpea Cowpea Common sun¯ ower Peanut Others Cracked maize Sorghum stalks Cornmeal Polished rice grains Cracked mungbean Sorghum leaves a Conidia

21.0 ad 21.0 a 12.9 ab 10.6 b 4.2 bcd 3.8 bcd 3.3 cd 0.8 d 0.4 d 0.4 d 0.4 d 0.4 d 0.3 d 0.1 d 5.5 bc 3.2 d 2.4 cd 1.9 cd 0.8 d 0.6 d

1

substrate ( 3 10 4 )

NUVb

DiVerencec

127.8 a 45.3 c 58.5 b 60.5 b 6.8 ef 6.1 ef 4.4 ef 1.1 e 0.8 e 0.8 e 0.8 e 0.6 e 0.5 e 0.2 e

** ** ** ** ns ns ns ns ns ns ns ns ns ns

9.1 ef 1.2 e 23.7 d 18.5 de 1.5 e 1.2 e

ns ns ** ** ns ns

were counted 4 WAI. Results are from pooled experiments.

5 constant light (400 l Em - 2 s - 1 PAR), NUV 5 constant near-ultraviolet light. c** 5 Signi® cant at 1% level and ns 5 not signi® cant. bL

d In a column, means having common letters are not signi® cantly diVerent according to Tukey’s test (P< 0.005).

404


TABLE 2.

EVects of incubation period, light, and temperature on production of A. alternata f. sp. sphenocleae conidia on sorghum seedsa Number of conidia g -

1

substrate ( 3 10 5 )

24ë C Light conditions Dd L L/D DiVerencef

3 WAIb

28ë C 4 WAI

0.7 ce 5.4 a 2.3 b

2.0 bc 6.2 a 2.3 c ns

3 WAI

4 WAI

DiVerencec

21.0 a 22.7 a 0.6 b

23.7 a 23.8 a 8.4 b

** ** *

*

a Results

are from pooled experiments. weeks after inoculation. in the number of conidia between 24ë C and 28ë C when conidia were collected at 3 WAI and 4 WAI; ** 5 signi® cant at 1% level, * 5 signi® cant at 5% level. dD 5 constant dark, L 5 constant light (400 l Em - 2 s - 1 ), L/D 5 12 h of alternating light and dark. e In a column, means having common letters are not signi® cantly diVerent according to Tukey’s test (P< 0.05). f DiVerence between the numbers of conidia between 3 WAI and 4 WAI under the diVerent light treatments; * 5 signi® cant at 5% level, ns 5 not signi® cant. b WAI

5

c DiVerence

light. Conidial production in the rest of the treatments was not signi® cantly (P < 0.05) aVected by NUV exposure in comparison to constant light. EVect of Temperature, Light Conditions and Incubation Period on the Number and Virulence of Conidia Produced on Sorghum Seeds There was a signi® cant interaction (P < 0.001) between temperature, light conditions and incubatio n period with respect to conidial production on sorghum seeds. Conidial production (0 to 1.5 3 10 4 g - 1 substrate) was not signi® cantly diVerent (P < 0.05) at 1 and 2 WAI under the diVerent temperature and light conditions (0± 1.5 3 10 4 g - 1 substrate). As in the previous experiment, none or very few conidia (0± 1.0 3 10 3 g - 1 substrate) were produced after 3 and 4 WAI at 32ë C under all light conditions. At 24ë C and 3 and 4 WAI, signi® cantly (P < 0.05) more conidia were produced under continuous light than under either dark or 12 h of alternating light and dark conditions (Table 2). In addition, there was no signi® cant diVerence between the production of conidia at 3 and 4 WAI. Most conidia were produced at 28ë C under either continuou s dark or continuou s light at 3 and 4 WAI. Conidial production at 28ë C was signi® cantly (P < 0.05) higher at 4 WAI than at 3 WAI. Overall conidial production was signi® cantly greater at 28ë C than at 24ë C. There was also a signi® cant (P < 0.001) interaction between temperature, light condition and incubatio n period with respect to the virulence of conidia produced on sorghum seeds. At 24ë C and 28ë C, the most virulent conidia at 3 WAI were produced in continuou s dark condition (Table 3). At 4 WAI the most virulent conidia were produced under either continuou s dark or light conditions. In addition, conidia collected at 4 WAI were signi® cantly (P < 0.05) more virulent than those at 3 WAI and conidia produced at 28ë C were more virulent than those produced at 24ë C. Conidia produced under 12 h of alternating light dark exposure showed the least virulence. EVect of Moisture Content and Quantity of Sorghum Seeds on Conidial Production There was a signi® cant interaction (P < 0.001) between the quantity of sorghum seeds and the volume of water added for conidia production, 1 : 1 ratio of seeds to water volume maximized conidial production (Table 4). The rate of conidia production g - 1 substrate remained constant as the quantity of substrate was increased. Low moisture content (26.6%) restricted growth and sporulation , whereas high moisture content (93.0%) caused the

405


TABLE 3.

EVects of incubation period, light, and temperature on virulence of A. alternata f. sp. sphenocleae conidia produced on sorghum seedsa Dry weight reduction (%)b 24ë C

Light conditions De L L/D DiVerenceg

28ë C

3 WAIc

4 WAI

3 WAI

4 WAI

DiVerenced

70.1 af 65.4 b 40.2 c

91.1 a 93.1 a 86.9 b

73.3 a 70.4 b 62.2 c

100.0 a 100.0 a 94.3 b

* * *

*

**

a Results

are from pooled experiments. expressed as reduction (%) in target plant dry weight 14 DAI. 5 weeks after inoculation. d DiVerence in dry weight reduction between 24ë C and 28ë C when conidia were collected at 3 WAI and 4 WAI; signi® cant at 5% level. eD 5 constant dark, L 5 constant light (400 l Em - 2 s - 1 ), L/D 5 12 h of alternating light and dark. f In a column, means having common letters are not signi® cantly diVerent according to Tukey’s test (P< 0.05). g DiVerence in dry weight reduction between 3 WAI and 4 WAI under the diVerent light treatments; ** 5 signi® cant at 1% level, * 5 signi® cant at 5% level. b Virulence c WAI

TABLE 4.

In¯ uence of sorghum seeds quantity and moisture content on production of A. alternata f. sp. sphenocleae conidiaa Number of conidia ( 3 10 5 g -

Sorghum seed weight (g) 10 20 30

1

)

Amount of water added (ml) 10 1.46 ab

(55.4) c

0.09 b (34.8) 0.07 b (26.6)

20

30

1.12 b (76.1) 1.60 a (54.5) 1.05 b (43.0)

0.98 b (93.0) 1.18 b (66.8) 1.48 a (54.1)

a Cultures were incubated 250 ml Erlenmeyer ¯ asks at 28ë C in the light for 4 weeks. Results are from pooled experiments. b In a column, means having common letters are not signi® cantly diVerent according to Tukey’s test (P< 0.05). c Values in parentheses are % moisture content of sorghum seeds after autoclaving.

substrate to become sticky and to aggregate, resulting in extensive mycelial growth, but limited conidial production. EVect of Storage Period on the Number, Germination and Virulence of Conidia Produced on Sorghum Seeds The number, germination and virulence of conidia all signi® cantly (P < 0.01) decreased during storage for 12 months under room conditions (246 2ë C) (Figure 1). However, even after 12 months of storage, there were more than 1.0 3 10 5 conidia g - 1 substrate with conidia germination and virulence of 85 and 78%, respectively. Comparison of Production Methods With Respect to Germination and Virulence of Conidia The production methods employed had no signi® cant eVect on conidia germination but did signi® cantly (P < 0.001) aVect conidia virulence (Table 5). Although conidia produced with the diVerent production methods were morphologicall y similar, sorghum seeds produced more chlamydospore s than 12 PDA or VJA. The most virulent conidia, as determined from eVects on target plant dry weight, were produced on sorghum seeds (98% weight reduction), whereas conidia from VJA were the least virulent (66% weight reduction).

406

FIGURE 1.


EVect of storage period on the number (A), germination (B), and virulence (C) of A. alternata f. sp. sphenocleae conidia produced on sorghum seeds.

407


TABLE 5.

In¯ uence of substrate on the number, germination and virulence of A. alternata f. sp. sphenocleae conidiaa

Substrate b 1 2

PDA VJA Sorghum seeds

Conidia /plate or ¯ ask ( 3 10 6 )

Germinationc (%)

Dry weight reductiond (%)

1.3e 0.7 18.5

98.3 af 96.5 a 97.2 a

86 bf 66 c 98 a

a Cultures were incubated at 28ë C in the dark for 4 weeks. Results are from pooled experiments. b 1 PDA 5 half-strength potato dextrose agar, VJA 5 V-8 juice agar. 2 c Germination of conidia was assessed after 8 h incubation at 24ë C in the dark. d Virulence expressed as reduction (%) on target plant dry weight 14 DAI. e Values in this column cannot be compared. f In these columns, means having common letters within the column are not signi® cantly diVerent according to Tukey’s test (P< 0.05).

DISCUSSION Alternaria species are well adapted to natural conditions with daily ¯ uctuations in temperature and light, but there is considerable variabilit y in the requirements for sporulatio n of Alternaria species in culture (Rotem, 1994). Light requirements are very distinct for the various Alternaria species (Leach, 1967). Photosporogenesi s in many Alternaria spp. consists of two distinctive phases: the inductive phase, leading to the formation of conidiophores ; and the terminal phase, leading to the formation of conidia (Aragaki et al., 1973). The inductive phase is stimulated by NUV light and the terminal phase proceeds best in darkness and is often inhibited by light. Although agar culture media systems are not feasible for the large-scale production of conidia, valuabl e knowledge can be attained on conidiation optimization from agar culture media experiments. Nutrition, temperature, light conditions and moisture aVected conidiatio n of A. alternata f. sp. sphenocleae on various agar media (Masangkay et al., 2000). Maximum production of virulent A. alternata f. sp. sphenocleae conidia was obtained on 12 PDA at 28ë C under constant NUV incubated for four weeks, or in 3± 4 days when using the S-media technique at 18ë C. Conidiophor e induction occurred on nutrient rich media and was stimulated by NUV. Meanwhile, formation of conidia proceeds best in darkness when nutrients are depleted under warm dry conditions or cool moist conditions (Masangkay et al., 2000). Requirements for sporulation on solid substrates are similar to those on standard agar media. The results for temperature are the same with 28ë C being optimal. Exposure to constant NUV stimulated sporulatio n on many solid substrates, but exposure to constant light or dark also produced abundan t conidia. The distinct morphogenetic stages during sporulation on agar media, where the initial development of conidiophore s followed by the formation of conidia, also occurred on solid substrates. Light inhibited the terminal phase on agar plates but not on the solid substrates, except perhaps on polished rice grains. As well as minimal light penetration to the bottom of the ¯ asks, much of the hyphal mass would be enclosed within the sorghum grains, and thus were protected from the inhibitor y eVect of light on sporulation . Of interest is the increase in production between the light and the NUV treatment. This increase was 10-fold on polished rice grains, while the increase on sorghum was only 5-fold, suggesting that perhaps more light penetrated into the white coloured rice grains, inhibitin g conidia formation. Conversely, agar cultures in plastic Petri dishes were uniformly exposed to constant light. Shaking the ¯ asks up to 14 DAI may also have aVected conidial production by creating a new physical condition that enhanced conidiophor e and/or conidia formation in light. Exposure to 12 h of alternating light and

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dark produced extensive mycelial growth compared with exposure to constant light or dark, the same response being observed with agar culture. Inhibition of vegetative development by wounding, starving or desiccation also can trigger sporulation of Alternaria species (Rotem, 1994). Colonizatio n of the sorghum grains would result in depletion of nutrients and moisture, producing conditions conducive to conidia formation, as was observed in both dark and light environments. Conidia production from a single ¯ ask containing 20 g of sorghum seeds with 54% moisture content (at the time of inoculation ) was 1.85 3 10 7 conidia, which was equal to the conidial production of approximatel y 14 and 26 plates of 12 PDA and VJA, respectively. Although the diVerent methods did not aVect conidia germinabilit y, conidia produced from sorghum seeds were more virulent than conidia from 12 PDA or VJA. Conidia produced on sorghum seed may have higher inherent energy levels or, since the virulence of A. alternata f. sp. sphenocleae is associated with phytotoxin(s ) (Masangkay et al., 1999b), conidial suspensions prepared from the sorghum seeds may have had higher phytotoxin concentrations than those prepared from the agar media. Compared to liquid fermentation systems, solid substrate fermentation has limitations. It is time-consuming and labour-intensive , diYculties may be encountered in harvesting conidia, and diYculties in preventing contaminatio n are common (Connick et al., 1990; Boyette et al., 1991). These constraints, however, may be minimized as economic parameters and technologies change. The biphasic methodology developed by Walker (1980, 1982) for A. cassiae has been attempted for A. alternata f. sp. sphenocleae, but was not particularly successful (Mabbaya d et al., unpublishe d data). Solid substrate fermentation with agricultural products has been used to produce inoculum of several bioherbicides (Morin et al., 1989; Connick et al., 1990; Zhang & Watson, 1997) and, as demonstrated here, is also feasible for A. alternata f. sp. sphenocleae. Solid substrates with a relatively low protein content, such as seeds of barley, millet, oat, sorghum, durum and hard red spring wheat, cracked maize, and polished rice grain encouraged sporulation, with sorghum, hard red spring wheat and barley producing most conidia. This was likely due to their large surface area and structure retention, lack of particle aggregation , and appropriat e nutrient and moisture content, which all contributed to restricting vegetative mycelial growth and to producing large numbers of conidia. Sorghum seeds were chosen for further evaluation because they are more readily availabl e at minimal cost in the Philippine s than hard red spring wheat or barley seeds. Solid-state production of A. alternata f. sp. sphenocleae on sorghum seed could be a feasible method for use in developin g countries to produce conidia at the village level or as a cottage industry. Improvements in substrate preparation , including sterilization and downstream processing are researchable activities, and methodologie s from insect/fungi biocontrol may be adapted. Presently, this biocontrol `product’ can be mass-produced on a readily availabl e substrate, in a process that does not require specialized equipment or specialized facilities. The inoculum is produced in a simple culture vessel, incubated in the dark at 28ë C, has a shelf-life of over a year, and can be applie d as a conidial suspension or as a granular material.

ACKNOWLEDGEMENTS This work was conducted under a memorandum of agreement between the International Rice Research Institute (IRRI) and McGill University. The work was funded in part by the United Nations Development Programme (UNDP) grant no. GLO/91/001/A /01/42 to IRRI, the Canadian International Development Agency CGIAR Linkage Fund, and the Natural Sciences and Engineering Research Council of Canada (NSERC) Operating Grants to AKW and SGH.


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