Short communication
Control of
Athelia rolfsii
disease on lentil seedlings using
6-pentyl-
a
-pyrone
S.L. Dodd
a,c,*, R.A. Hill
b, A. Stewart
ca
School of Biological Sciences, P.O. Box 92 017, University of Auckland, Auckland, New Zealand
b
Horticulture and Food Research Institute of NZ, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand
c
Soil Plant and Ecological Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand
Accepted 6 December 1999
Abstract
The volatile fungal metabolite 6-pentyl-a-pyrone (6PAP) suppressed Athelia rolfsiidisease of lentil seeds and seedlings when applied onto the surface of the soil, on 13 mm diameter Whatman assay disks. The metabolite had no phytotoxic eects on lentil. This is the ®rst study to show that 6PAP is active in the soil against the vegetable pathogenA. rolfsii. 72000 Elsevier Science Ltd. All rights reserved.
Keywords:6-Pentyl-a-pyrone;Trichoderma;Athelia rolfsii; Biological control; Antibiosis
The volatile fungal metabolite 6-pentyl-a-pyrone (6PAP) is produced by a number of Trichoderma species and is thought to play a role in the biological control activity of this fungus against plant pathogens (Dennis and Webster, 1971; Cutler et al., 1986; Ghisal-berti and Sivasithamparam, 1991; Graeme-Cook and Faull, 1991). In this report, we assess the ability of dif-fering amounts of 6PAP to suppress infection of lentil seeds and seedlings by Athelia rolfsii (Curzi) Tu and Kimbrough (= Sclerotium rolfsii Sacc.), a soilborne pathogen of numerous commercial vegetable crops.
This assay was performed in enclosed tissue culture pots (TC290SP, Almed Lily NZ Ltd, Auckland, NZ) containing 15 g of soil (Smiths Horticultural Distribu-tors Ltd, Auckland, NZ) which had been sieved through a 2 mm2 wire mesh, autoclaved twice for 1 h at 1218C (15 p.s.i.) on consecutive days and incubated at room temperature in the dark for 2 weeks prior to use. Within each pot, the soil was overlaid onto
ap-proximately 20 ml of 1.5% (w/v) water agar to help maintain high moisture levels. Individual tissue culture pots were subsequently sown with six lentil seeds around the perimeter and, where appropriate, three sclerotia of A. rolfsii were placed on the surface of the soil in the centre of the pot. Prior to sowing, seeds were surface sterilised by immersion in 1% sodium hypochlorite for 10 min followed by immersion in ster-ile distilled water ®ve times for 10 min at a time. Scler-otia used to inoculate pots were harvested from 28 day-old colonies grown on potato dextrose agar at 378C. To aid germination, sclerotia were rinsed in ster-ile distilled water and twice blotted dry on sterster-ile ®lter paper.
The 6PAP (Aldrich Chemical Company Inc., Mil-waukee, WI, USA) metabolite was introduced to pots on an assay disk. Sterile Whatman assay disks (13 mm diameter1 mm thick) were treated with either 0, 1, 5
or 10 mg 6PAP dissolved in 1 ml of absolute ethanol and applied in a series of ten 100 ml applications, allowing the ethanol to evaporate between appli-cations. Each disk was then cut into four pieces and placed evenly around the pot between the seeds and sclerotia. A negative control was included which had
Soil Biology & Biochemistry 32 (2000) 1033±1034
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* Corresponding authors at Soil Plant and Ecological Sciences Div-ision, P.O. Box 84, Lincoln University, Canterbury, N2. Tel.: +64-3-3252811; fax: +64-3-3253843.
neither sclerotia or 6PAP added. Also included was a treatment of 10 mg of 6PAP in the absence of A. rolf-sii sclerotia to determine any phytotoxic eects of 6PAP. Additional sterile distilled water was added to each pot prior to incubation at 278C under ¯uorescent lights. Following 10 days incubation period, disease symptoms were scored using the numerical scale of: healthy (0), small lesion at the base of the stem (1), girdling lesion (2), and completely necrotic seedling (3). There were ®ve pots per treatment. Mean and standard errors of the disease symptom scores were calculated for each treatment. The entire assay was performed twice and each trial analysed separately. For each treatment, mean disease index and standard errors were calculated from the ®ve pots.
Greatest disease suppression was achieved with the 10 mg 6PAP treatment per pot, giving a mean disease index of 1.0 and 0.8 for trials one and two respectively, compared with an index of 18 for the relevant patho-gen controls. The 5 mg 6PAP treatment also sup-pressed disease with an index of 14.8 in trial one and 6.6 in trial two, whereas the 1 mg 6PAP treatment had no eect on disease in either trial. When tested for phytotoxicity, the 10 mg treatment alone had no detri-mental eect on the plants in either trial (Table 1).
The levels of 6PAP used in this study were not able to inhibit the germination of A. rolfsii sclerotia when disks were placed between 6 and 12 mm from them. In contrast, mycelial growth was restricted to between 3 and 5 mm in length in the 10 mg 6PAP treatment, compared to the 0 mg control treatment where myce-lial growth exceeded the edge of the pots, growing up the sides.
This is the ®rst study to show that 6PAP is active in
sterile soil against the vegetable pathogen A. rolfsii and not phytotoxic to lentil. Being a volatile com-pound, it is not feasible to add 6PAP directly to pot-ting mix as a protectant. However, adding a fungus that produces the metabolite during the growth of a plant is highly desirable. Trichoderma spp. are now being added to commercial potting mixes as a condi-tioner to provide protection and, in some instances, enhance plant growth (John Hunt at Agrimm Technol-ogies Ltd. Christchurch, NZ, pers comm.).
It is common practice to use agar plate assays to screen large numbers of organisms for their ability to produce antibiotics with biocontrol activity. Such assays are rapid and economical to perform as opposed to larger scale, labour intensive glasshouse and ®eld trials. However, we have observed that such assays do not necessarily re¯ect metabolite activity in the glasshouse or ®eld (Alison Stewart pers comm.). We propose that the bioassay described in this study provides an alternative, fast and easy assay for asses-sing the antibiotic activity of volatile fungal metab-olites on soilborne plant pathogens capable of mycelial growth through the soil. The soil assay has the added advantage of being able to assess the disease suppres-sion activity of such metabolites, as well as their eects on pathogen germination and mycelial growth. A soil based assay should give a more accurate prediction of metabolite activity in a glasshouse. The use of non-sterile agricultural soil would more accurately predict the ®eld environment. Subsequent ®eld trials involving Armillaria spp. on kiwifruit vines con®rm the activity of 6PAP against soilborne pathogens in the ®eld (Robert Hill pers comm.; Cutler and Hill, 1994).
References
Cutler, H.G., Cox, R.H., Crumley, F.G., Cole, P.D., 1986.
6-Pentyl-a-pyrone from Trichoderma harzianum: its plant growth inhibi-tory and antimicrobial properties. Agricultural and Biological Chemistry 50, 2943±2945.
Cutler, H.G., Hill, R.A., 1994. Natural fungicides and their delivery systems as alternatives to synthetics. In: Wilson, C.L., Wisniewski, M.E. (Eds.), Biological Control of Post-harvest Diseases. Theory and Practice. CRC Press, Boca Raton, Florida, pp. 135±152.
Dennis, C., Webster, J., 1971. Antagonistic properties of species-groups of Trichoderma. II. Production of volatile antibiotics. Transactions of the British Mycological Society 57, 41±48. Ghisalberti, E.L., Sivasithamparam, K., 1991. Antifungal antibiotics
produced byTrichoderma spp. Soil Biology and Biochemistry 23, 1011±1020.
Graeme-Cook, K.A., Faull, J.L., 1991. Eect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic pro-duction on selected pathogens in vitro. Canadian Journal of Microbiology 37, 659±664.
Table 1
Eect of 6PAP concentration on Athelia rolfsii infection of lentil seedlings
Treatment Mean disease indexd2SE
Trial one Trial two
aPots were inoculated with three sclerotia ofAthelia rolfsiiand an assay disk treated with ethanol only.
bPots were inoculated with an assay disk treated with ethanol only.
c
Pots were inoculated with an assay disk containing 10 mg of 6PAP only.
d
Mean disease index calculated from the total disease index of ®ve pots.
S.L. Dodd et al. / Soil Biology & Biochemistry 32 (2000) 1033±1034
