Acremonium mycoherbicide for biocontrol of dandelion

Spores of a biocontrol fungus, Acremonium sp. ATCC 74368, have been found to function well as a biocontrol agent in a method for biocontrol of dandelion (Taraxacum officinale).

BACKGROUND OF THE INVENTION 
The present invention relates generally to biocontrol fungi and more 
specifically to fungi useful in controlling dandelions. Biological control 
of many plant species using microorganisms and compounds isolated 
therefrom is known. In U.S. Pat. No. 4,755,207, phytotoxic Alternaria 
cassiae spores were formulated and applied to sicklepod. In U.S. Pat. No. 
4,209,826, Amphbotrys ricini was used successfully as a mycoherbicide 
against texasweed. In U.S. Pat. No. 4,606,751, Bipolaris sorghicola spores 
were demonstrated to kill johnson grass. 
Certain unsuccessful efforts have been undertaken to identify and obtain 
biological agents, notably fungal phytopathogens, having phytotoxic 
activity against dandelions. Dandelions, classified as Taraxacum 
officinale, are common and persistent broadleaf weeds with wide geographic 
range. Dandelions are listed as the most important turf weeds in the U.S. 
in more states (42) than any other dicotyledon. To date, dandelions have 
been uncontrolled under typical environmental conditions except by 
mechanical means or with chemical herbicides which are disfavored because 
of their toxicity to humans and animals and to beneficial plants. The most 
common chemical herbicide used to control dandelions is the fungicide 
2,4-D, which has been shown to contain dioxins in certain formulations and 
which may be a human carcinogen. Other known chemical herbicides effective 
against dandelions include Basagran and Round-Up. 
Several biocontrol agents have been ineffective against dandelion growth. 
For example, in U.S. Pat. No. 4,390,360, an Alternaria cassiae fungus 
selected for biological control of a number of plant weeds was 
demonstrated to be ineffective against dandelions. Likewise, U.S. Pat. No. 
4,929,270 discloses that compounds isolated from a fungal pathogen of 
spotted knapweed were selectively phytotoxic against knapweed, but spared 
dandelions and a number of other dicots and monocots. The effective target 
range of most mycoherbicides is narrow and, to date, biological control of 
dandelions has not been readily accomplished. 
A mutated strain of S. sclerotiorum for controlling dandelions has been 
patented. Riddle, G.E., et al., Weed Science 39:109-118 (1991), showed in 
certain isolates of Sclerotinia sclerotiorum and S. minor a positive 
correlation between virulence on dandelion leaves and reduction in amount 
of dandelion foliage or number of dandelion plants. However, S. 
sclerotiorum is a soil-borne fungus having a different host range than the 
fungus of the present invention. 
Other fungal isolates have been described which show activity against 
dandelions under conditions of extremely high humidity not typically 
encountered in temperate climates. 
What is desired is a biological agent that is toxic to dandelions under 
typical environmental conditions but is non-toxic to humans, animals and 
desired plants. 
BRIEF SUMMARY OF THE INVENTION 
The present invention is summarized in that an Acremonium fungus that can 
been cultured as a substantially pure preparation exhibits good control of 
dandelion growth. The Acremonium fungus, Acremonium spp., AM 18, deposited 
with the American Type Culture Collection, 12301 Parklawn Drive, 
Rockville, Md. 20852, and accorded ATCC accession number 74368, is 
characterized and identified below. 
The present invention is also directed toward a method for controlling 
dandelions using the novel fungus. 
It is an object of the present invention to provide a phytotoxic biocontrol 
agent that effectively controls dandelion growth. 
It is another object of the present invention to effectively control 
dandelion growth without using non-biological chemical agents. 
It is an advantage of the present invention that the use of non-biological 
chemical agents is avoided. 
It is a further advantage of the present invention that the phytotoxic 
fungus identified herein can be 100% effective at relatively low dosages 
to control dandelions and appears to have no toxicity against other plants 
or animals. 
It is another advantage of the present invention that the fungus is readily 
reproduced and stored for subsequent use. 
Other objects, features and advantages of the present invention will become 
apparent from the following specification. 
DETAILED DESCRIPTION OF THE INVENTION 
A novel fungal strain of Acremonium that exerts biological control over 
dandelion plant species was isolated from diseased dandelions and was 
grown and maintained in substantially pure cultures. Acremonium spp., AM 
18 (ATCC 74368) was one of several fungi isolated from severely diseased 
dandelions in Howard County, Maryland. The symptoms of the diseased 
dandelions included leaf lesions and severe stem necrosis. The fungus 
sporulates abundantly in culture on PJA or DYA agar plates, producing 
numerous small conidia. This fungus can also be grown to produce conidia 
in liquid culture using simple substrates. The strain has been deposited 
in the American Type Culture Collection under the designation ATCC 74368 
and shall hereinafter be referred to as "Acremonium sp. ATCC 74368" or 
simply as "ATCC 74368". 
A "substantially pure" culture shall be deemed a culture of a fungus 
containing no other fungal species in quantities sufficient to interfere 
with replication of the desired fungus. "Biological control" or 
"biocontrol" is defined as control of dandelion plant growth by the use of 
a second organism. "Biological control" of dandelions shall be deemed to 
exist when a statistically significant increase in plant disease or 
mortality occurs after an effective quantity of the substantially pure 
culture is applied to the treated plant. A change in plant disease or 
mortality is measured on the scale of 0-5 described below in connection 
with the plant disease or mortality assay. An "effective quantity" shall 
be that quantity of the fungus sufficient to result in a significant 
increase in disease or mortality in treated plants, measured in the plant 
disease and mortality assay. Plants, including seeds, seedlings, and 
mature plants, that respond to an effective quantity of a fungus or toxin 
shall be referred to as "susceptible" to the fungus or toxin. Clearly, if 
no quantity of a fungus or any toxin or other compound is an effective 
quantity as so defined, that fungus, toxin, or compound is not capable of 
exerting biological control over the dandelion. 
It is envisioned that certain mutants of ATCC 74368 can also be isolated 
that also provide biological control comparable or superior to that 
provided by ATCC 74368. Phytotoxic mutants of ATCC 74368 can include both 
naturally occurring and artificially induced mutants. Other such 
controlling mutants of ATCC 74368 can be artificially induced by 
subjecting ATCC 74368 to the mutagen, N-methyl-nitrosoguanidine in 
conventional ways. ATCC 74368 and those mutants capable of exerting such 
biological control shall sometimes be referred to collectively as 
"phytotoxic" fungi. Any mutant that retains effective biocontrol 
properties is within the scope of the present invention. It is likely that 
other such phytotoxic Acremonium strains that exert biological control 
over dandelions, as well as members of related genera such as 
Cephalosporium, will be isolated. 
It is further envisioned that the fungus or mutants thereof may be 
genetically altered by the addition or removal of genes that affect fungal 
growth. In particular, for example, it is believed that introduction into 
the fungus of genetic material that confers resistance to an herbicide or 
to a biocontrol agent will make the fungus an excellent candidate for 
co-delivery with such an herbicidal agent to a cultivated area. In that 
case, the herbicide would be effective against other undesirable weeds and 
the fungus would be effective against the dandelion. 
It is contemplated that the phytotoxic activity for each individual 
pathogenic fungus varies over a significant range. Accordingly, the amount 
of a particular fungus necessary to control dandelion growth can be 
determined empirically by in vitro studies of the type described herein. 
Based on such studies, an "effective amount" can readily be determined for 
a particular treating organism. 
The biological effect of the fungus on the dandelion plant appears to be 
systemic. It is envisioned that the fungus is itself a parasite, or 
produces a phytotoxin having biocontrol activity. If one or more 
phytotoxins exist, they will be prepared in isolation from the phytotoxic 
fungi and will be characterized chemically so as to be identifiable 
independent of its source in cultures of ATCC 74368, or phytotoxic mutants 
thereof. It is specifically envisioned that any phytotoxin or phytotoxins 
responsible for biocontrol of dandelions are not unique to ATCC 74368 or 
its phytotoxic mutants, and, therefore, that a wide variety of other 
fungal organisms that do not derive from ATCC 74368 but which contain such 
a phytotoxin or phytotoxins could also be isolated. A phytotoxin could be 
isolated from ATCC 74368 or from its controlling mutants or other fungus 
by filtering the fungi from the culture media in which they have been 
grown. Alternatively, the phytotoxin could be isolated from the fungi 
rather than from their culture media, if the toxin is not secreted. Other 
conventional purification and concentration steps may then be undertaken 
as may be considered convenient or desirable, so long as the toxin remains 
active, as may be demonstrated by the plant disease and mortality assay 
(described below) that was initially developed to test plant protection 
activity observed using ATCC 74368. It is believed that the same assay can 
be used to test other candidate fungal strains for biocontrol activity. 
The phytotoxin, if any, may readily be isolated from biocontrolling fungi 
which produce them by recovering the supernatant from sporulating colonies 
of the microorganisms. The supernatant, as described above, can be 
fractionated in a column and then separated by electrophoresis to identify 
the fractions which exhibit the biocontrol activity. Using high voltage 
paper electrophoresis, or comparable purification technique, the bioactive 
molecules can readily be repeatably recovered. 
Based upon the observed biocontrol properties of the fungus, the following 
is a disclosure of a suitable plant disease and mortality assay whereby a 
test material such as a fungus, a phytotoxin, an extract or the like, may 
be tested for its ability to exert biological control over a dandelion. 
The seed of the dandelion plant to be controlled is planted in a planting 
medium. The planting medium can be vermiculite in water or any other 
planting medium in which the seed will grow and develop to an age of four 
weeks or less. The test material is sprayed onto the plant to runoff at a 
concentration of between 10.sup.6 and 10.sup.7 conidia/ml. The plant 
should be kept initially for at least 12 hours with free moisture and then 
maintained for up to one week at a temperature between 24.degree. and 
28.degree. C. before evaluating disease and mortality as described above. 
Control plants sprayed only with water should be included in the assay. 
Mortality of plants treated with an effective fungal agent will be readily 
apparent when compared to controls, the growth of which should be 
unimpeded under these conditions. It is understood that an equally valid 
test for biocontrol may be performed on uncultivated dandelion plants 
growing naturally, taking into consideration the guidance of this 
specification as to temperature, dew period and plant age. 
The following is a suitable disease rating scale for use in conjunction 
with the assay: 
0=no disease 
1=1-25% leaf necrosis 
2=26-50% leaf necrosis 
3=51-75% leaf necrosis 
4=76-99% leaf necrosis 
5=dead plant. 
A fungal isolate (or phytotoxin isolated therefrom, or structurally-related 
phytotoxin) effective for biocontrol of Taraxacum officinale can be 
formulated as a biocontrol agent as follows. The fungus can be added to an 
inert carrier such as bentonite, silica, or alumina, or to a nutritive 
agent such as bran, corn cobs, newspaper, sawdust, or the like. 
Combinations of nutrients and inert carriers are preferred. The 
formulation can be granular (for broadcast application) or sprayable (in 
connection with a hose-end or pump sprayer). Liquid formulations 
containing the fungus comparable to formulations prepared for other 
biocontrol agents may also be used. The formulation can vary, in ways 
known to the art, depending upon whether the fungus is to be used 
preventively or curatively. 
ATCC 74368, its phytotoxic mutants, other strains that exert biological 
control over dandelions, inocula containing ATCC 74368, its phytotoxic 
mutants or other biocontrolling strains, or one or more phytotoxins 
produced by, isolated from or structurally identical or equivalent 
thereto, and combinations thereof are all intended to fall within the 
scope of the present invention. Likewise, methods for preparing biocontrol 
agents, and methods for exerting biological control on dandelion growth, 
which employ ATCC 74368,its phytotoxic mutants, other biocontrolling 
strains, inocula containing ATCC 74368 or its mutants, or one or more 
phytotoxins produced by, isolated from or structurally identical or 
equivalent thereto, or combinations thereof are also within the scope of 
the present invention. 
It is also envisioned that ATCC 74368, as well as a phytotoxin or 
phytotoxins present in ATCC 74368, may have comparable biocontrol activity 
against certain other related broadleaf weed plants, including other 
dandelion species. The fungus has no apparent biocontrol activity against 
grasses. 
The invention will be more fully appreciated upon consideration of the 
following non-limiting Examples.

EXAMPLES 
Fungi were collected in Howard County, Maryland from severely diseased 
flowering dandelions and from plants with seedheads. The symptoms of the 
diseased dandelions included leaf lesions and severe stem necrosis. 
Isolations were made to potato-dextrose agar (PDA) with kanamycin. 
Organisms recovered included Colletotrichum, Alternaria, Fusarium, 
Epicoccum, Cladosporium, a cleistothecial and a perithecial fungus, a 
yeast, and an Acremonium. 
To determine whether any of the isolates had phytotoxic activity, 
dandelions with seedheads growing outside were drenched with a mixture of 
the Acremonium, the yeast, and the Colletotrichum, each at about 
1.times.10.sup.6 spores per ml. After about five days, the dandelions were 
severely diseased. Fungi recovered from the diseased dandelions included 
the Acremonium, Alternaria, Epicoccum, Fusarium and Cladosporium. 
Next, six 4.times.6 inch trays were potted with Redi-earth and were seeded 
with dandelions. After about 3 weeks, plants were inoculated with either 
the Colletotrichum at 1.times.10.sup.6 spores/ml or the Acremonium at 
about 1.times.10.sup.7 spores/ml. Both spore suspensions were from 
PDA+kanamycin plates that had been incubated at room temperature with no 
specific light requirements for 12 days. The spores were rinsed from an 
actively growing culture using distilled water. The spore concentration 
was determined using a hemocytometer. A total of 35 ml of inoculant were 
used for each fungus. Two flats, each containing 5 dandelion plants 
(approximately 3 weeks old) (for a total of 10 treatments) were also 
inoculated for each treatment. Two control flats were sprayed only with 
water. After inoculation, plants were incubated at 24.degree.-25.degree. 
C. with a 12 hour photoperiod and a 24 hour dew period. The Acremonium 
infected all tested plants, killed 40% of the plants, and caused 
significant damage to those that survived. The damaging effects included 
severe stem necrosis, leaf curling, leaf lesions, stunted growth and 
death. Plants inoculated with the Colletotrichum were not killed, but 
leaves did develop lesions. 
Two separate Acremonium isolates (#1429 and #1430) and the yeast thus 
obtained were more rigorously assessed for their ability to exert 
biological control against dandelion. The yeast isolate was soon 
thereafter determined to be non-pathogenic and was not evaluated further. 
All cultures were maintained in cryogenic storage (-80.degree. C.). 
Subcultures were made directly from the cryogenic cultures for each 
experiment after it was determined that virulence declined and was lost 
after several culture transfers on agar medium. Isolates 1429 and 1430 
caused significant plant mortality on 3-4 week old dandelion plants within 
one week of inoculation. In early experiments, most plants were killed 
within 48 hours. Although both isolates were determined to be equally 
pathogenic, isolate 1429 was used in all subsequent studies. 
In these experiments, dandelion was seeded directly into plastic pots 
containing vermiculite and was grown for 3-4 weeks (to the 4-6 leaf stage) 
in lighted growth chambers at 28.degree. C. (14 hour photoperiod). Each 
pot was thinned to 3-6 plants per pot after plant emergence. Plants were 
examined daily and were rated for disease seven days after treatment. Each 
plant was rated separately and the average rating for each pot was used 
for comparison in this plant disease and mortality assay. The disease 
rating scale was as noted above. 
Dew period and dew temperature can influence plant infection. To determine 
the influence of these factors, conidia were harvested from 4-5 day old 
PDA plates and were standardized at 1.times.10.sup.6 conidia/ml using a 
hemocytometer. Plants were sprayed to runoff and placed immediately into a 
dark dew chamber at 28.degree. C. for 24 hours unless indicated otherwise. 
To determine the dew period requirement, plants were placed into a dew 
chamber for 0, 4, 8, 12, 16, 20, or 24 hours at 28.degree. C. Results of 
these tests are shown in Table 1. At least approximately 12 hours of free 
moisture were required for effective weed control using ATCC 74368. After 
8 hours, plants showed moderate levels of leaf necrosis but plant 
mortality was limited. 
TABLE 1 
______________________________________ 
Influence of dew period on infection 
and disease development 
Dew Period (hr) 
Disease Rating (0-5) 
Mortality (%) 
______________________________________ 
0 0.8 0 
4 0.6 0 
8 2.4 18 
12 4.5 72 
16 4.9 90 
20 4.9 97 
24 4.9 97 
______________________________________ 
To determine the influence of dew period temperature, pots were incubated 
for 24 hours in separate dew chambers adjusted to 16.degree., 20.degree., 
24.degree., 28.degree., 32.degree. or 36.degree. C. Following incubation 
in the dew chamber, pots were placed into lighted growth chambers at 
28.degree. C. (14 Hour photoperiod) for the remainder of the experiment. 
Each experimental treatment included four pot replicates. Controls 
included one or more pots sprayed with water. As is reported in Table 2, 
significant disease development and plant mortality occurred at dew 
temperatures between 24.degree. and 28.degree. C. Disease development was 
limited at 20.degree. and 32.degree. C. Significant plant mortality was 
observed at 36.degree. C., but this was attributed to heat stress rather 
than to any fungal phytotoxicity. 
Several attempts were made to determine the influence of post-inoculation 
temperature on disease development. However, susceptible plants were 
killed so rapidly under disease-conducive conditions that this proved 
difficult to determine and the experiments were discontinued. 
TABLE 2 
______________________________________ 
Influence of dew temperature on infection 
and disease development 
Dew Temperature 
Disease Rating 
Mortality 
(hr) (0-5) (%) 
______________________________________ 
16 1.4 10 
20 1.8 4 
24 4.4 63 
28 4.9 95 
32 1.1 0 
36 3.2 35 
______________________________________ 
To determine the preferred inoculation dose, various concentrations of 
conidia in the range of 10.sup.4 to 10.sup.7 were prepared by serial 
dilution and were inoculated to runoff onto susceptible four-week old 
plants under disease conducive conditions of temperature and dew period. 
Plants were placed into a dew chamber at 28.degree. C. (14 hour 
photoperiod). Each experimental treatment included five pot replicates. 
Plants were rated for disease seven days after inoculation. At a 
concentration of 10.sup.6 or greater, excellent control of dandelion 
plants was observed. At concentrations of 10.sup.5 or lower, disease 
development was limited and no mortality was observed, as is shown in 
Table 3. 
TABLE 3 
______________________________________ 
Influence of conidial concentration on infection 
and disease development 
Disease Rating 
Mortality 
Conidial Concentration 
(0-5) (%) 
______________________________________ 
10.sup.4 0 0 
10.sup.5 1.0 0 
10.sup.6 5.0 100 
10.sup.7 5.0 100 
______________________________________ 
To determine the influence of plant age on infection and disease severity, 
five pot replicates were seeded at weekly intervals for six weeks. Plants 
were sprayed to runoff with a conidial suspension of 1.times.10.sup.6 
conidia/ml when plants had reached ages of 2, 3, 4, 5, 6, and 7 weeks. In 
general, plants four weeks old (with 4-6 true leaves) or less were highly 
susceptible to the pathogen and were readily killed under conditions 
favorable for infection and disease development. Although numerous 
pinpoint lesions developed on older plants and older infected leaves 
collapsed, plants usually were not killed and grew out of the infection. 
This experiment was repeated several times with similar results although 
the exact cutoff point between plants with significant mortality and those 
without varied somewhat depending upon experimental conditions. 
TABLE 4 
______________________________________ 
Influence of plant age on susceptibility 
Plant Age Disease Rating 
Mortality 
(wk) (0-5) (%) 
______________________________________ 
2 5.0 100 
3 5.0 100 
4 3.0 90 
5 2.0 0 
6 1.0 0 
7 1.0 0 
______________________________________ 
In summary, excellent biological control of young dandelion plants was 
obtained within seven days of inoculation of ATCC 74368 under 
disease-conducive conditions. Mortality was often observed within 48 
hours. These mortality results are comparable or superior to chemical weed 
control agents. Susceptible plants were rapidly killed following at least 
12 hours of free moisture at temperatures between 24.degree. and 
28.degree. C. Temperatures between 28.degree. and 32.degree. C. may also 
be effective. These temperatures are normally encountered during the time 
period in which dandelion requires treatment. A 12 hour free moisture 
requirement for infection is common among plant pathogenic fungi. Although 
the moisture requirement is not seen to be a limitation on the use of the 
biocontrol agent, since irrigation is often available for turfgrass, it is 
known in the art that addition of as little as 1-5% vegetable oil or other 
adjuvant can reduce the free moisture requirement by four hours or more. 
The present invention is not intended to be limited to the embodiments and 
examples presented herein, but rather to encompass all such modifications 
and variations as come within the scope of the appended claims.