Biological control of weeds using AAL-toxin

It has been discovered that AAL-toxin, a known host-specific phytotoxin produced by Alternaria alternate f. Sp. lycopersici, has a broad range as a pre-emergent or post-emergent bioherbicide. A method using AAL-toxin has been developed for controlling certain weeds, including duckweeds, jimsonweed, black nightshade, prickly sida, redroot pigweed and northern jointvetch. This phytotoxin can be used pure, as a cell-free filtrate, a crude filtrate, or a crude suspension of the culture.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the biological control of several weed 
species by the use of a pathogenic toxin from a strain of the fungus 
Alternaria alternate. 
2. Description of the Prior Art 
Members of the genus Alternaria are known to produce a wide range of 
phytotoxic compounds which affect a large number of the plants on which 
the fungus is found (Bruce, V.R., Stack, M.E., and Mislivec, P.B. [1984] 
J. Food Sci. 49:1626-1627; Harvan, D.J., and Pero, R.W. [1976] J.V. 
Rodricks, ed. Advances in Chemistry, Series 149. pp 344-355). These 
phytotoxins include alternariol, alternariol monomethylether (AME), 
altenuene, altenuic acid, tenuazonic acid (TA), tentoxin, alternaric acid, 
AK-toxin, and AAL-toxin, and possess a broad range of biological 
activities and metabolic effects (Bruce et al., supra; Harvan et al., 
supra; Nishimura, S., and Kohmoto,K. [1983] Annual Rev. Phytopathology 
21:87-116). 
These phytotoxins have been referred to as `host-specific` because they 
"are toxic only to the host that is susceptible to the pathogen which 
produces the toxin, and if they induce nearly all symptoms of the disease 
are considered to be definitive chemical probes in the study of disease 
susceptibility and physiological stress at the molecular level" (Bottinii 
A.T., and Gilchrist, D.G. [1981] Tetrahedron lett. 22:2719-2722). 
"Physiological, biochemical, genetic, and histological data all confirm 
that these toxic compounds are the key determinants of disease and of host 
selection by the producing fungi" (Scheffer, R.P. [1989] Kohmoto, K. and 
Durbin, R.D., ed. Host-Specific Toxins, pp. 1-17). 
Furthermore, it is believed by phytopathologists that tolerance and 
sensitivity to a toxin is controlled by the same genes in the same way 
that they control resistance and susceptibility to the fungus (Scheffer, 
Robert P. and Livingston, Robert S. [1984] Science 223:17-21). If a fungus 
does not grow on a plant, the phytotoxin produced by the fungus will not 
affect that plant, depending on whether or not it has dominant or 
recessive alleles (Grogan, R.G., Kimble, K.A., and Misaghi, I. [1975] 
Phytopathology 65:880-886). In general, resistance in cultivare is 
equivalent to insensitivity to a metabolite released by the pathogen 
(Scheffer, R.P., supra). It has been suggested that these host-specific 
toxins are suitable for use as tools for screening resistant genotypes in 
plant breeding programs (Clouse, S.D., and Gilchrist, D.G. [1987] 
Phytopathilogy 77:80-82; Clouse, S.D., Martensen, A.N. and Gilchrist, D.G. 
[1985] Journal of Chromatography 350:255-263; Scheffer et al., supra). 
Even in a single species there may be a variety of strains which will be 
morphologically the same but can produce different toxins to which 
different hosts are susceptible. These are called pathotypes. A. alternata 
includes many pathotypes which are disease-producing in specific plants 
(Nishimura et al., supra; Stierle, A.C., Cardellina II, J.H., and Strobel, 
G.A. [1989] J. Natural Products 52:42-27). 
One of these pathotypes is A. alternate f. sp. lycopersici which causes a 
serious stem canker disease affecting the leaves, stems, and fruits of 
susceptible tomato cultivars. However, the infection is unpredictable in 
that there are other tomato varieties which are unaffected by either the 
fungus or its toxin. 
The structure of a host-specific phytotoxin responsible for stem canker 
disease has been elucidated. It was shown to be a long-chain 
ninhydrin-positive polyol called AAL-toxin (or Al-toxin) (Bottini, A.T., 
Bowen, J.R., and Gilchrist, D.G. [1981] Tetrahedron lett. 22:2723-2726; 
Bottini, A.T., and Gilchrist, D.G. supra). It has also been shown that in 
tomato cultivars which are susceptible to AAL-toxin, the pair of alleles 
involved is at the asc locus. The plants had three significantly different 
levels of toxin sensitivity, which were inherited as an incomplete 
dominant and corresponded to the genotype at the asc locus (Clouse et al. 
[1987] supra). 
Apparently, however, not all stem canker disease is caused by AAL-toxin. A 
stem canker causing phytotoxin isolated from an A. alternata f. sp. 
lycoversici phytotype has been reported which did not react with ninhydrin 
indicating the absence of primary or aromatic amines. The toxin was 
effective on EarlyPak Tomatoes, but not on jimsonweed (Datura stramonium) 
or other solanaceous species tested (Gilchrist, D.G. and Grogan, R.G. 
[1975] Phytopathology 66:165-171). 
There are many fungi which are pathogenic to weeds and which produce 
phytotoxins that could be useful as herbicides (Abbas, H.K., Boyette, 
C.D., Hoagland, R.E., and Vesonder, R.F., [1991] Weed Sci. 
39:673-677;Boyette, C.D. [1986] Plant Sci. 45:223-228; Boyette, C.D., 
Weidemann, G.J., Te BeeBt, D.O. and Quimby, Jr., P.C., [1991] Weed Science 
39:678-681); Stierle, et al., supra). Fusarium spp. are particularly 
plentiful throughout the world. An isolate of Fusarium moniliforme, 
obtained from infected jimsonweed, was found to produce fumonisin 
phytotoxin (Abbas, et al., supra). Fumonisin, while structurally similar 
to AAL-toxin, is obtained from a species known to have a broad host 
spectrum, whereas A. alternata f. sp. lycoversici is host-specific. 
SUMMARY OF THE INVENTION 
In view of the art described above, we were surprised to discover a method 
of controlling weeds of the type including duckweeds, jimsonweed, black 
nightshade, redroot pigweed, northern jointvetch, and prickly sida 
comprising applying to the weeds a phytotoxic amount of AAL-toxin produced 
by the fungus A. alternata. The AAL-toxin can be applied to the weeds in 
any suitable form including as cultures of the A. alternata fungus in 
water. Both a fungus-infected corn culture and a fungus-infested rice 
culture are likewise suitable. The AAL-toxin can be applied as a 
post-emergent or pre-emergent herbicide.

DETAILED DESCRIPTION OF THE INVENTION 
A. alternata is easily isolated from susceptible tomato plants exhibiting 
symptoms of stem canker disease by known procedures (Abbas, H. K., supra). 
Isolates can be grown on potato-dextrose agar, malt agar and hay infusion 
agar and identified on the basis of conidial morphology (Ellis, M.B. 
[1971] Dematiaceous Hyphomycetes pp.464-466). 
To produce AAL-toxin, the fungus is cultured on any suitable medium such as 
yellow corn kernels or converted long grain enriched rice (Abbas, H. K., 
supra). The AAL-toxin as identified is described below. 
The bioherbicidal compositions of the invention are prepared by dispersing 
the cultures in suitable medium at application rate of active agent 
preferably ranging from about 0.1 to about 2.0 Kg/hectare. Suitable media 
include inert solid, powder, or granular, dry materials or liquid 
materials. Water is a particularly suitable material for dispersing 
AAL-toxin-containing cultures. The compositions can include non-inert 
material such as fertilizers, herbicides and the like. Suitable 
concentrations of the toxin can be determined easily by those skilled in 
the art as will become clear from the examples, but is preferably in the 
range of about 0.0001% to about 99.9% by weight. As a practical matter, it 
is suitable to use formulations of AAL-toxin from crude fungal inocula or 
fractions thereof, such as cell-free filtrates, thereby obviating the need 
to isolate the pure compound. However, formulations of the pure compound 
are certainly suitable. 
The phytotoxic amount (i.e. that amount needed to kill the weed) of 
AAL-toxin in each formulation can be determined easily for each target 
weed species by anyone skilled in the art. 
A preferred method of applying AAL-toxin containing formulations is by 
spraying pout-emergent weeds or by spraying soil before the weeds emerge. 
Other methods of application will be obvious to those skilled in the art. 
The following examples are intended only to further illustrate that which 
the inventors believe to be their invention and should not be taken as 
limiting the present invention in any way. 
Example 1 
Nine Alternaria alternata and two Cladosporium cladasporioides isolates 
were obtained from infected `Beefsteak` tomato plants exhibiting symptoms 
of stem canker disease. The isolates were grown on potato-dextrose agar 
(PDA), malt agar and hay infusion agar and stock cultures of these were 
maintained in the SWSL fungal repository on PDA and in the NRRL/ARS 
Culture Collection, Peoria, Ill. One A. alternata f. sp. lycopersici was 
provided by D.G. Gilchrist, Dept. of Plant Pathology, University of 
California, Davis, Calif., as A. alternata AS 27-32P. This isolate was 
used in these studies as a positive control because it is known to produce 
AAL-toxin, and is reported to cause stem canker in certain varieties of 
tomatoes. The isolates were given the designation of A. alternata SWSL 1, 
2, 3, 5, 6, 8, 10, 11, and 12, C. cladasporioides SWSL 7 and 9, and A. 
alternata AS 27-32P was designated as SWSL 4. 
Corn meal agar (CMA) cultures (12 to 14 days old) of isolates SWSL 1-12 
were homogenized at 22.degree. to 24.degree. C. in 50 mL sterilized 
distilled water. This inoculum contained conidia and mycelium at a 
propagule density of 2.times.10.sup.7 mL.sup.-1. 
A second inoculum was produced by growing the isolates on autoclaved, 
converted long-grain enriched rice. The fungus and rice were incubated for 
28 days at 24.degree. to 26.degree. C. at 35 to 37% moisture content. The 
fungus-infested rice was transferred to screen-bottomed trays and allowed 
to air-dry at room temperature 22.degree. to 26.degree. C. for 72 to 96 
hours in a ventilated hood. Two hundred grams of fungus infested rice were 
ground into a fine powder. Twenty grams of the fungus-infested rice powder 
were added to 100 mL of distilled water, stirred for 1 to 2 minutes, 
sonicated for 15 minutes and then sieved through double cheese cloth. The 
resulting preparation consisted of spores and mycelium at a propagule 
density ranging from 1 to 10.sup.4 mL.sup.-1 as determined by 
hemacytometer counts. 
Standard samples of alternariol, alternariol monomethyl ether (AME) and 
tenuazonic acid (TA) were obtained commercially. Tentoxin was provided by 
S.O. Duke [USDA-ARS, Southern Weed Science Laboratory (SWSL), Stoneville, 
Miss.]. AAL-toxin was purified from a crude filtrate of A. alternata f. 
sp. lycopersici grown on liquid media. Pure AAL-toxin was used at 
concentrations of 0.2 mg/ml distilled water; TA at concentrations of 25 
mg/60 mL of 10% DMSO; and AME at concentrations of 20 mg/60 mL 10% DMSO. 
Crude filtrates were prepared by homogenizing 40 g of fungus-infested rice 
in 200 mL distilled water and filtering through a double layer of cheese 
cloth. Half of this filtrate was taken to produce cell-free filtrate by 
filtration through 0.45 .mu.m Millipore filters. For the phytotoxin test, 
100 g of fungus-infested rice were extracted successively with hexane (300 
mi), dichloromethane (2x, 300 mL) and 60% (v/v) aqueous methanol (300 mL). 
AME was detected in the dichloromethane extract by thin layer 
chromatography (TLC) on silica-gel plates (E.Merck, Darmstadt, West 
Germany) developed in the solvent system chloroform:methanol [95:5 (v/v)] 
versus AME standards. AME was isolated by silica-gel column chromatography 
at a concentration of 58 mg per 100 g of fungus-infested rice. Identity of 
AME was confirmed by comparison of its NMR, UV, and IR spectra with 
authentic samples. TA and AAL-toxin were detected on C.sub.18 reverse 
phase TLC plates in the solvent system methanol:water [75:25 (v/v)] versus 
authentic samples. These two toxins were isolated by C.sub.18 reverse 
phase chromatography using methanol:water [65:35 (v/v)]. Their identities 
were confirmed by co-chromatographing each metabolite with authentic 
samples. TA was further characterized by its IR and UV spectra. The 
fungus-infested rice extracts of isolates containing AME, TA and AAL-toxin 
were analyzed for tentoxin and alternariol. Neither one was detected. 
Only the extracts of SWSL 1 were found to contain the following phytotoxins 
at the indicated concentrations: AAL-toxin (100 .mu.g/g rice media); 
tenuazonic acid (TA) [10, .mu.g/g]; and alternariol monomethyl ether (AME) 
[580 .mu.g/g] (Table 1). Tentoxin and alternariol were not detected in the 
fungus-infested rice extracts of any of these isolates. The extract of 
SWSL 4 was found to contain the phytotoxin AAL-toxin at a rate of 85, 
.mu.g per g of fungus infested rice. 
TABLE 1 
______________________________________ 
Production of Phytotoxin by 
Alternaria alternata grown on rice. 
Concentration 
.mu.g for g fungus- 
Phytotoxin infected rice 
______________________________________ 
AAL-toxin 100 
Tenuazonic acid (TA) 10 
Alternariol monomethyl ether (AME) 
580 
Alternariol ND 
Tentoxin ND 
______________________________________ 
Pathogenicity tests on jimsonweed: Jimsonweed seeds were mechanically 
scarified with sandpaper and planted in a commercial potting mixture 
supplemented with a slow release fertilizer (N:P:K 14:14:14), contained in 
peat strips (12.times.5.5 cm.sup.2 pots/strip). The plants were watered as 
needed, and the greenhouse temperature was maintained between 28.degree. 
and 32.degree. C. with 40 to 60% relative humidity. The photoperiod was 
ca. 14 h at ca. 1600 to 1899 uE.m..sup.-2 s.sup.-1 at midday. The fungal 
inoculum from each isolate was applied with an atomizer to run-off. 
Control groups received a filtrate of autoclaved rice or distilled water. 
Jimsonweed plants, 1 and 2 wks old (2- to 4 leaf stage) were used in these 
experiments. Following inoculation, plants were incubated on greenhouse 
benches under conditions as described above. Three replicates of 12 plants 
each were used for each treatment. The experiment was repeated twice. 
Symptom development was monitored daily. Heights of six Jimsonweed plants 
were measured at the beginning and the end of the experiments. Dry weights 
of plant material above the soil were determined at the end of the 
experiments after drying for 48 hr at 60.degree. to 70.degree. C. The 
results are shown in Table 2. 
In intact plants, the damage resulting from the crude, cell-free filtrates 
and the AAL-toxin was identical, including various sizes of necrotic spots 
on the leaves and stems of intact plants. The growth and dry weight of 
plant material biomass were affected similarly by the cell-free filtrate, 
AAL-toxin and crude filtrate. The reduction in biomass was 68% and 46% for 
AAL-toxin and cell-free filtrate, respectively. Plant height was reduced 
by cell-free filtrates and AAL-toxin significantly, as compared to control 
groups. DMSO (10% v/v) incited identical damage as AME and TA in DMSO. 
This indicates that DMSO was responsible for the damage to the leaves of 
intact plants. The two C. cladasporioides isolates (SWSL 7 and SWSL 9) 
from the same infected tomato plants were neither pathogenic nor did they 
produce any detectable phytotoxins. They were therefore used as controls 
for the method. 
The crude filtrates of isolates SWSL 4 and SWSL 1 each contained AAL-toxin 
which caused similar damage to jimsonweed plants. 
SWSL 1 was deposited in the NRRL/ARS Culture Collection, Peoria, Ill. and 
was designated as NRRL#18822. 
TABLE 2 
______________________________________ 
Effects of various Alternaria alternata 
isolates and various phytotoxins on 
the growth of 2-wk old jimsonweed. 
Absolute Plant Dry 
Fungus or amt. of Height.sup.a 
Weight.sup.b 
Phytotoxins 
Code toxins change reduction 
(Source) no. used (mg) (cm) (%) 
______________________________________ 
Alternaria 
SWSL 1 2 (AAL) 11.6 31 
alternata 2 ND 16.7 12 
(Tomato 3 ND 18.3 5 
plants) 5 ND 16.3 12 
6 ND 16.3 1 
8 ND 16.9 1 
10 ND 18.3 9 
11 ND 18.3 5 
12 ND 18.3 1 
Alternaria 
SWSL 4 1.6 (AAL) 12.0 27 
alternata 
(AS 27-32P) 
(Pure 
culture) 
Cladosporium 
cladasporioides 
(Tomato SWSL 7 ND 17.0 1 
plants) 9 ND 17.0 1 
Control H.sub.2 O -- 17.4 1 
Crude- SWSL 1 2 (AAL) 8.7 44 
filtrate 
(Rice-infested 
fungus) 
Cell-free SWSL 1 1.8 (AAL) 9.9 46 
filtrate 
(Rice-infested 
fungus) 
AAL-toxin SWSL 1 4 3.4 68 
Tenuazonic 
TA 25 8.1 45 
acid 
Alternariol 
AME 20 7.6 45 
monomethyl 
ether 
Dimethyl DMSO 60 mL 7.0 43 
sulfoxide 
Controls H.sub.2 O 60 mL 16.4 2 
______________________________________ 
.sup.a Means of six plants +/- standard deviation 
.sup.b The mean of 3 replicates (each of 12 plants) 
+/- standard deviation 
ND = not detected 
Spores from either isolate grown on CMA applied to jimsonweed at a rate of 
2.times.10.sup.7 spores/ml, with or without dew period did not produce any 
symptoms. 
Example 2 
Excised jimsonweed leaves were used to test the biological activities of 
crude and cell-free filtrates and secondary metabolites. Excised leaves 
were placed on moistened filter paper inside 9-cm diameter sterile petri 
plates. The inocula of crude filtrates, cell-free filtrates, and the 
phytotoxin standards were applied to the leaves with micropipets. Amounts 
used were 100 .mu.l to adaxial or abaxial surfaces at concentrations of: 
(a) 20 g/100 mL distilled water for the crude and cell-free filtrates; (b) 
25 mg of TA dissolved in 60 mL of 10% DMSO; (c) 35 mg of AME dissolved in 
60 mL of 10% DMSO; and (d) AAL-toxin 0.2 mg mL.sup.-1 distilled water. Six 
leaves were used for each treatment. Control leaves received either rice 
filtrate, distilled water, or 10% (v/v) DMSO. The plates were sealed with 
parafilm and incubated under continuous or 12 h light (20 uE.m..sup.-2 
s.sup.-1). The phytotoxic effects on the treated excised leaves were 
evaluated visually for damage for 14 days. Crude and cell-free filtrates 
and AAL-toxin caused similar damage to excised leaves, characterized by 
autolysis diffusing from the point of treatment along the veins adaxially 
or abaxially to leaves. AME and TA caused no visible damage to excised 
leaves after 10 days, while the 10% DMSO solution caused 
moderate-to-severe necrosis. After 12 days, TA-treated plants exhibited 
chlorotic halos around the treatment points. These phytotoxins all 
required high concentrations (&gt;1000 .mu.g/mL) to be effective. Results are 
shown in Table 3. 
TABLE 3 
______________________________________ 
Effects of fungal filtrates and secondary metabolites 
produced by A. alternata on excised leaves of jimsonweed.* 
Fungal filtrates 
Code Conc. Amt./Material 
Phyto- 
or toxins No. (.mu.g/mL) 
(mg) leaf toxicity 
______________________________________ 
Control H.sub.2 O 100% 0.10 mL. - 
Crude-filtrate 
SWSL #1 200 2.00 + 
(Rice-infested 
fungus) 
Cell-free SWSL #1 200 2.00 + 
filtrate 
(Rice-infested 
fungus) 
AAL-toxin SWSL #1 0.2 0.02 + 
Tenuazonic 
TA 0.42 0.04 + 
acid** 
Alternariol 
AME 0.58 0.06 + 
monomethyl 
ether 
Dimethyl- DMSO 10% 0.10 mL - 
sulfoxide 
______________________________________ 
*Six leaves were used for each treatment. The phytotoxic damages were 
evaluated visually for 14 days. 
- = no phytotoxic effects 
+ = phytotoxic effects 
**Leaves showed chlorotic halos around the treatment points after 12 days 
 
Example 3 
AAL-toxin was applied to excised jimsonweed and black nightshade (Solanum 
nigrum L.) true leaves to determine a dose-response curve. Primary and 
secondary leaves from greenhouse-grown 20-day-old jimsonweed plants were 
used in this study. The primary and secondary leaves were obtained from 
20-day-old black nightshade plants (seeds were purchased from Thompson 
Seed Co., Fresno, Calif.). The AAL-toxin used in this study was produced 
and purified in the following manner. 
A. Growth of cultures: 
Two hundred grams of Uncle Ben's converted long-grain enriched parboiled 
rice (Uncle Ben's, Inc., Houston, Tex.) were placed with 120 mL of 
distilled water into one liter flasks and were allowed to stand about one 
hour until the water was absorbed. The flasks were shaken to uniformly 
distribute the moist substrates, closed with tight cotton stoppers and 
autoclaved 60 min at 15 lbs pressure. Immediately after autoclaving, the 
flasks were shaken vigorously to break up clumps. They were allowed to 
stand 24 hr and the autoclaving and shaking process was repeated. The 
flasks were then inoculated as soon as they were cool, using isolates of 
Alternaria alternata NRRL 18822, and maintained in stock cultures on corn 
meal agar slants in small vials. To recover the fungus, a small piece of 
inoculum was placed on PDA for 7 to 10 days. One to two cm.sup.2 inoculum 
was removed and added to autoclaved rice. The flasks were incubated in the 
incubator at 28.degree. C. for 4 wks with daily shaking for the first few 
days to permit the fungus to penetrate the rice uniformly. Shaking was 
discontinued after complete invasion approximately 5-7 days after 
inoculation. The fungus and rice were stored for 4 wks in the incubator 12 
h light and 12 h dark. The fungus-infested rice was transferred to a 
screen-bottomed tray, was allowed to air dry in a ventilated hood, was 
ground to the consistency of flour, and used on these studies (biological 
and chemical). 
B. Extraction: 
One kg of fungus-infested rice was soaked in chloroform overnight at a 
ratio of 1 gram fungus-infested rice: 5 mL of chloroform. This was placed 
in a blender for 5 min at high speed. The chloroform layers were 
discarded. The residue which contained AAL-toxin was transferred to a 
screenbottomed tray and was allowed to air dry in a ventilated hood. The 
dried residue was soaked in H.sub.2 O:methanol (60:40) overnight, at a 
ratio of 1 gram fungus-infested rice per 5 mL of extracting solvent. This 
was placed in a blender for 5 min at high speed. The filtrate was 
collected by centrifugation. The extracting process was repeated 2 times 
with residue as described above. The filtrates were combined and the 
methanol was removed by rotary evaporator. The AAL-toxin is in the water 
layers. 
C. Purification: 
This process was patterned after fumonisin purification as described in 
detail by Vesonder, R.F., R.E. Peterson, R.D. Plattner, and D. Weisleder. 
[1990] Mycotoxin Res. 6:85-88. Briefly, the water aliquot was passed 
through an Amberlite XAD-2 (Sigma Chemical Co.) column. The Amberlite 
XAD-2 column was washed successively with distilled water and methanol. 
The methanol eluate contained AAL-toxin as evidenced on TLC c.sub.18 
reverse-phase plate with F.sub.254 s fluorescent indicator (EM Science, 
Cherry Hill, N.J.) versus an authentic sample developed in the solvent 
system methanol:water (75:25). The AAL-toxin contained in the methanol 
eluate was purified by flash column chromatography on a 50 gram 
reverse-phase C.sub.18 packing (Water Associates, Milford, Mass.). The 
column was eluted with 60% aqueous methanol, and 10 mL fractions 
collected. A white solid was obtained in the fractions containing 
AAL-toxin. Twenty milligrains of 95% pure AAL-toxin was obtained from 430 
mg of crude toxin contained in the methanol eluate from the XAD-2 column. 
The AAL-toxin identity was confirmed by FAB-mass spectrometry (M+peak at 
522). 
AAL-toxin (14.2 mg) was dissolved in 71 mL sterile distilled water to yield 
a 200 .mu.g/ml solution. This stock solution was diluted serially by two 
with equal amounts distilled water to a concentration of 0.01 .mu.g/ml (10 
ng/mL). 
Six leaves were used for each treatment. Control leaves received distilled 
water. Excised leaves were placed on moistened filter paper inside 9-cm 
sterile Petri plates. The different concentrations of AAL-toxins were 
applied to the leaves with a 10 .mu.l micropipette, 4 to 12 times per 
leaf, depending on its size. The absolute amount of each application was 
determined. The plates were incubated under 14 h light (498 
.mu.E.m..sup.-2 s.sup.-1) in the growth chamber at 25.degree. C. with 
80-85% R.H. Observations of phytotoxicity were made at frequent intervals 
for five days. The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Effects of AAL-toxin on jimsonweed 
and black nightshade leaves. 
Toxin Absolute amt. of 
Phytotoxicity 
concentration 
toxin/drop Jimson- Black 
(.mu.g/mL) 
(ng) weed nightshade 
______________________________________ 
0 0 - - 
0.01 0.1 - + 
0.02 0.2 - + 
0.05 0.5 - + 
0.10 1 - + 
0.20 2 - ++ 
0.39 3.9 - ++ 
0.78 7.8 - ++ 
1.56 15.6 + +++ 
3.13 31.3 + +++ 
6.25 62.5 + +++ 
12.5 125 ++ +++ 
25 250 ++ +++ 
50 500 +++ +++ 
100 1000 +++ +++ 
200 2000 +++ +++ 
______________________________________ 
Six leaves were used for each treatment. Control groups received distille 
water. Leaves were incubated under 14 hours light (498 .mu.E..sup.-1 
m..sup.-2 s..sup.-1) in growth chamber at 25.degree. C. for five days. 
Absolute amount of AALtoxin per drop (each 10 .mu.L) was calculated based 
on volume and concentration of the pure toxin. 
- = No phytotoxicity observed 
+ = Less than 1/3 of leaf autolyzed 
++ = Less than 1/2 of leaf autolyzed 
+++ = More than 1/2 of leaf autolyzed 
Example 4 
Thirty-one weed and cultivated plants from 10 families were used. They 
ranged in age from seven to ten days old at the time of spraying. Seeds of 
plants used in these experiments were obtained from commercial companies 
or collected locally. The number of plants of each cultivar varied between 
ten to fifty per pot depending on the plant species. Seeds of each 
cultivar were planted in a vermiculite potting mixture as described 
previously. The experiment was confirmed by repeating twice. One 
concentration of AAL-toxin at 0.2 mg/ml was prepared in 50 mL distilled 
water. Filtrate of fungus-infested rice was prepared by homogenizing 20 g 
in 100 mL distilled water and filtering through a double layer of cheese 
cloth. An aerosol sprayer was used to spray the AAL-toxin solutions and A. 
alternata filtrates on the plants until run off. Plants were kept in the 
greenhouse under the same conditions as described in Example 1. Symptoms 
were observed daily until the end (last two weeks) of the experiment and 
included chlorosis, necrosis, stunting and mortality. The results are 
shown in Table 5. 
TABLE 5 
______________________________________ 
Response of various crop and weed species 
tested for susceptibility to AAL-toxin 
and Alternaria alternata NRRL 18822. 
FAMILY Disease 
Common name, scientific name, cultivar 
reaction.sup.a 
______________________________________ 
AMARANTHACEAE 
Redroot pigweed, ( Amaranthus retroflexus L.) 
S-5 
COMPOSITAE 
Cocklebur (Xanthium strumarium L.) 
S-2 
CONVOLVULAVEAE 
Morningglory (Ipomoea wrightii Gray) 
I 
CUCURBITACEAE 
Cucumber (Cucumis sativus L.) 
S-2 
GERANIACAEA 
Wild geranium (Geranium dissectum L.) `cutleaf` 
S-3 
`Carolina` S-2 
GRAMINEAE 
Barley (Hordeum vulgare L.) 
I 
Bermuda grass [Cynodon dactylon (L.) pers.] 
I 
Corn (Zea mays L.) `Truckers Favorite` 
I 
Johnsongrass [Sorghum halepense (L.) Pers.] 
S-1 
Oats (Avena sativa L.) I 
Rice (Oryza sativa L.) S-1 
Grain sorghum [Sorghum bicolor (l.) Moench] 
I 
`Texas C-124` 
Wheat (Triticum aestivum L.) 
I 
LEGUMINOSAE 
Alfalfa (Medicago sativa L.) 
S-3 
Crimson clover (Trifolium incarnatum L.) 
S-3 
Sicklepod (Cassia obtusifolia L.) 
S-2 
Hemp sesbania (Sesb S-4 
ania exalta (Raf.) Cory.) 
Northern jointvetch [Aeschynomene virginica 
S-5 
(L.) B.S.P.] 
American jointvetch (Aeschynomene americna L.) 
S-1 
Indian jointvetch (Aeschynomene indica L.) 
S-2 
Soybean [Glycine max (L.) Merr.] 
`Forrest` S-1 
`Centennial` S-1 
LEMNACAEA 
Common duckweed (Lemna minor L.) 
S-5 
Duckweed (Lemna pausicostata L.) 
S-5 
MALVACEAE 
Cotton (Gossypium hirsutum L.) `Stoneville 213` 
S-1 
Prickly sida (Sida spinosa L.) 
S-5 
Velvetleaf (Abutilon theophrasti Medic.) 
S-1 
Spurred anoda [Anoda cristata (L.) Schlect) 
S-2 
SOLANACAEA 
Tomato (Lycopersicon esculentum Mill) 
`Beefsteak` S-2 
`Marion` S-2 
Jimsonweed (Datura stramonium L.) 
S-5 
______________________________________ 
.sup.a Reaction: 
S = susceptible, where 1 equals small (1 mm), nonenlarging lesions to 5 
equals plant death; 
I = immune.