USDA-ARS, Southern Weed Science Research Unit, Stoneville, Mississippi 38776, USA

Background and objectives
Whole, ripe tomatoes (Lycopersicon esculentum), as well as leaf discs of tomato, black nightshade (BNS) (Solanum nigrum), and jimsonweed (JW) (Datura stramonium), can be used as assays for the production of alternaric acid (AA) by isolates of Alternaria solani [1]. A. solani is the causative agent of early blight on tomatoes and produces several toxins including alternaric acid (AA), 10-deoxyalternaric acid (10-DAA), and solanopyrone-A (SP-A) [2]. The objective of this study was to compare the phytotoxicity of the above toxins on tomato, BNS and JW.

Materials and methods
Whole Ripe Tomato assay: whole ripe tomatoes were surface sterilized with ethanol and dried under a hood. A sterilized cork borer 2 mm2 inside diam. was used to make holes 2x2 mm in whole firm ripe tomatoes, 3 holes per tomato. 2 mm2 of A. solani inoculum was inserted in each hole, covered with parafilm and kept in a growth chamber at 22-25C for 25 days. A drop (5 ml) of 1 or 5 mM toxin was placed in each hole of the other tomatoes and the same procedure was followed as above. Control (untreated) and treated ripe tomatoes were observed frequently for 25 days and symptoms were recorded.
Leaf disc assay: Tomatoes (26 biotypes), JW and BNS were grown from seeds in the greenhouse. Twenty leaf discs, 2 mm inside diam., were placed in small petri dishes, 3.5 mm inside diam., containing sterilized water only as a control or toxin in water at 1 or 5 mM with three replicates and were incubated at 25C for 48 h. Cellular toxicity was determined by measuring conductivity of the bathing media with a conductivity meter at 0, 24 and 48 h. Chlorophyll loss was determined at 48 h by UV spectrometry. Chlorophyll was extracted after 48 h treatment and assayed by the method of Hiscox and Israelstam. Leaf discs were soaked for 24 h in darkness in 5 ml DMSO at room temperature. Sample tubes were then centrifuged at 500 g for 10 min and the spectrophotometric absorbance of the supernatant was measured at the appropriate wavelengths for determining total chlorophyll using Arnon's equations. A phytotoxicity rating scale was used to grade biotypes according to phytotoxicity as healthy (little phytotoxic effect), mild, severe, and very severe phytotoxic effects.

Result and conclusions
Small dark rots, approximately 2 cm (inside diameter), were produced in whole tomatoes inoculated with AA at 1 and 5 mM within 7 days, while the control fruits remained sound. Tomato leaf discs from 22 biotypes were used to compare the phytotoxicity of AA, 10-deoxyalternaric acid (10-DAA), and solanapyrone-A (SP-A) at 5 mM. Phytotoxic effects included an increase in conductivity which was determined by measuring the cellular leakage in bathing media with respect to control of chlorophyll loss. All biotypes were equally susceptible to AA at 5 mM. At 1 mM, 11 biotypes showed mild effects, while 11 showed severe effects, of which five were very severe. In leaf disc assays of BNS and JW, AA at 5 mM caused dramatic phytotoxic effects 48 h after the initial exposure to toxin. Lesser concentrations of AA, including 1 mM, also caused significant phytotoxicity. SP-A was less active than AA at 5 mM and minimally active at lower concentrations. 10-DAA had minimal effects at all concentrations.

These assays are useful tools for testing phytotoxic isolates of Alternaria solani and are simple and inexpensive. They also confirm that AA is a main metabolite responsible for the symptoms of early blight and can also be screened for by the same assays. 10-DAA and solanapyrone A do not cause detectible phytotoxicity in these tomato bioassays.

1. Abbas HK, Johnson BJ, 1997. Phytopathology 87, S2.
2. Tabuchi H, Hamamoto T, Miki S et al., 1994. Journal of Organic Chemistry 59, 4749-4759.