WO1993022923A1

WO1993022923A1 - Mycoherbicide

Info

Publication number: WO1993022923A1
Application number: PCT/AU1993/000229
Authority: WO
Inventors: Eric John Cother; Ross Leslie Gilbert
Original assignee: The Minister For Agriculture & Rural Affairs (For The State Of Nsw)
Priority date: 1992-05-19
Filing date: 1993-05-18
Publication date: 1993-11-25

Abstract

Weeds of the family Alismataceae in rice and other crops are controlled by applying to the weed an effective amount of a suspension of spores of the fungus Rhynchosporium alismatis in a suitable carrier.

Description

MYCOHERBICIDE Technical Field

The present invention is directed to a mycoherbicide and a method for the control and/or eradication of weeds from the Alismataceae family in crops, particularly rice (Oryza sativa ) crops and other situations wherever weeds of this family are a problem. Background Art

Weeds of the family Alismataceae are a problem particularly in man modified habitats such as water courses, drainage channels, ditches and particularly rice crops. There is currently no registered chemical which satisfactorily controls or eradicates these particular weeds, which can compete vigorously with commercial crops such as rice and will persist in the field for long periods.

Particularly, the weeds from the family Alismataceae which are currently a problem to the commercial rice industry include:- Alisma lanceolatum (Alisma)

A. plantago - aquatica (water plantain) Sagittaria montevidensis (Arrowhead) S . graminea

Damasonium minus (star fruit) These species and others in the Alismataceae family occur at varying degrees throughout the world and the relative problem of each varies from place to place.

The use of chemical control agents against other weeds, but which are ineffective against weeds of the Alismataceae family, has resulted in weeds of the

Alismataceae family becoming more predominant in the weed flora. Thus, weeds of the Alismataceae family are starting to emerge as a significant problem in many areas. There is no known effective or currently registered control agent for at least some of the Alismataceae weeds.

Also, of particular problem to the wild rice (Zizania palustris) industry in North America, are Alisma triviale (also referred to as A. plantago - aquatica ) and Sagittaria lati folia.

Wild rice is more sensitive than common rice to damage from currently available herbicides that might be applied for control of Alismataceae weeds. This makes the situation with wild rice even more difficult and increases the need for an alternative or complimentary form of weed control. Disclosure of the Invention

The present inventors have found that fungal spores of the fungus fiiiync-osporiuiπ alismatis (Oudem.) J J Davis, are effective in controlling and/or eradicating the weeds of the family Alismataceae, especially the particular weeds previously mentioned which are relevant to the rice industry.

In a first aspect the present invention consists of a mycoherbicide for controlling weeds of the family Alismataceae, comprising spores of Rhynchosporium alismatis, together with a suitable carrier therefor.

The fungus Rhynchosporium alismatis has been previously described in the literature. This fungus was previously known as Ascochyta alismatis . This fungus and reference to the current name is described by E. Punithalingam in Mycological Papers No 159 published by C.A.B. International Mycological Institute pages 157-162, issued 8 March 1988.A number of isolates of the fungus, isolated from Alismataceae plants, are lodged with and are available from the Australian National Collection of Plant Pathogenic Fungi (Herb. DAR) at the Biological & Chemical Research Institute, Rydalmere, New South Wales, Australia. A list of isolates lodged with Herb. DAR is set out hereunder as Table I. TABLE 1

Isolates of R . alismatis lodged at Herb. DAR

Accession No. Host

DAR

67515 A. lanceolatum

62345 A. lanceolatum

67517 A. lanceolatum

67514 A. lanceolatum 67509 A plantago-aquatica

67510 A plantago-aquatica 67508 A plantago-aquatica 67513 A plantago-aquatica

67512 A plantago-aquatica 67516 S .montevidensis

67511 D. minus

It is believed that fungi of the species R . alismatis only produce spores as reproductive propagules. However, to the extent that they may be induced to produce some other reproductive propagules, these are to be included within the term "spore" as used in this specification.

It has been found that the fungal spores of J?. alismatis may be applied directly to the weed plant, or preferably and more likely, the fungal spores are used as the active ingredient in a mycoherbicide for application to a whole crop by conventional methods, such as spraying. The fungus is suitable for use as a mycoherbicide because it:- i) is relatively easy to culture and it sporulates profusely in vitro; ii) causes debilitating disease in the target weeds; iii) is safe to the economically important crop in which the weed grows; iv) has a narrow host range; v) occurs naturally throughout most of the world, e.g., Australia, USA and Europe. It has been found that the weeds of the Alismataceae family are naturally susceptible to the fungus, which may be pathogenic to the weeds and which causes leaf and petiole lesions. It has been found that even a very small amount of the fungus, which may only result in a small number of lesions, is sufficient to control the weed to a point where it stunts the growth of the weed, which can then no longer compete to any extent, thus allowing the normal growth of a desired crop, such as rice. Thus, it is emphasized that, to be effective, it is not absolutely necessary that the fungus kills the weed, but rather, stunting of the growth of the weed and the reduction in competition for light/space/nutrients and decreased fecundity can be equally effective. This is consistent with findings elsewhere regarding the effect of mycoherbicides on plants. The fungal spores are preferably in an essentially axenic suspension in the carrier ie. the preparation contains substantially only propagules of R. alismatis and no other organisms or propagules of such organisms.

Preferably, the concentration of the fungal spores in the mycoherbicide is at least 8 x 10 spores/ml. Spore concentrations below about 8 x 10 spores/ml will also affect weeds of the family Alismataceae and in some cases concentrations even significantly below about 8 x 10 spores/ml will be considered sufficient to control those weeds in a commercial situation.

In another preferred embodiment the Rhynchosporium alismatis is selected from the group of isolates consisting of the isolates listed in Table I of the present specification. It is preferred that the spores are from the isolate DAR67515 which has been deposited with the Australian Government Analytical Laboratories as accession number N93/19114 under the Budapest Treaty on

3 May 1993.

The fungus can be grown on solid media or, for large scale production, liquid media are satisfactory. Optimal spore production has been achieved on lima bean agar or in an aqueous extract of lima beans.

In a second aspect the present invention consists of a method of controlling or eradicating weeds of the family Alismataceae, comprising applying to plants thereof an effective quantity of fungal spores of the fungus

Rhynchosporium alismatis .

In a third aspect the present invention consists of a method of selectively controlling or eradicating weeds of the family Alismataceae in a crop, water channel or the like, comprising applying to the crop, water channel or the like an effective quantity of fungal spores of the fungus Rhynchosporium alismatis .

In another preferred embodiment of the second and third aspects of the invention, the weeds are selected from the group comprising Alisma lanceolatum, A. canaliculatum, A. plantago - aquatica , Sagittaria montevidensis, S. graminea, S . latifolia, S . pygmaea and

Damasonium minus . In another preferred embodiment of the second and third aspects of the invention, the crop, pasture or the like is a rice crop.

In another preferred embodiment of the second and third aspects of the invention the fungal spores are applied to plants in the seedling stage.

In another preferred embodiment of the second and third aspects of the invention the fungal spores of

-R ynchosporiui-i alismatis are applied to the crop, water channel or the like in the form of a liquid suspension. Preferably, the liquid suspension containing the fungal spores is applied to a plant or seedling until excipient runoff of the liquid suspension occurs.

In another preferred embodiment of the second and third aspects of the invention the liquid suspension of Rhynchosporium alismatis fungal spores is applied to plants in a crop, water channel or the like until incipient runoff occurs on the plants. Best Mode of Carrying out the Invention

Hereinafter a preferred embodiment of the various aspects of the present invention will be described with reference to the following examples. The examples are in no way intended to be limiting in terms of the scope of the present invention but merely representative of a preferred mycoherbicide and method of controlling weeds of the family Alismataceae _r according to the present invention. In particular, it is emphasized that the concentration range of spores used is in no way limiting but are merely preferred concentrations. As stated previously, even small amounts of the fungal spores have been found to be sufficient to stunt the growth of the weed to an extent which will effectively control the growth and subsequent problem of the weed in a field situation. Also, the mycoherbicide will effectively function with any isolate of Rhynchosporium alismatis . It is likely that certain isolates of R. alismatis will differ in certain characteristics which will make their selection for inclusion in the mycoherbicide of the present invention particularly appropriate. Throughout the present specification, the expression "crop, water channel or the like" includes anywhere the weeds of the Alismataceae will grow, and particularly includes crops such as rice crops as well as irrigation channels and drainage channels. Production of Spores The fungus can be grown on solid media, optimum spore - 1 -

production being achieved in Difco^® Lima Bean Agar. For large scale production the following liquid medium is satisfactory:

Lima beans, lOOg are soaked overnight in 800 mis distilled water, boiled for 1 hr then strained to remove particulate matter. Make up filtrate to

1 litre.

Spore production is carried out in orbital shake flask culture. The medium is inoculated with spores or mycelial fragments and incubated on an orbital shaker at 25 C for 5 days with a 12 hr photoperiod.

Spores are harvested and concentrated by centrifuging and resuspended in 10% aqueous glycerol solution at a

7 concentration of 2 x 10 spores per ml. In one particular experiment flasks containing 125mls of culture media (see above) were inoculated with 20 x 3 mm dia agar plugs from a culture. Flasks were incubated on an orbital shaker at 140 rp , 25 C and 12 hour photoperiod provided by a mix of cool white and black lamps 35cm above the flasks. Spores were harvested by centrifuging for 2 mins at 3000 rpm. Counts were made from resuspended spores using a haemocytometer. Maximum spore production was achieved after 10 days incubation in full strength lima bean broth (refer Table 2).

This formulation is then spayed onto the plant to complete wetness using an air powered atomiser.

It is preferred that the application is made to very young seedlings to achieve maximum growth suppression.

Example 1

7 Three aqueous suspensions containing 2.37 x 10 spores/ml of Rhynchosporium alismatis, were prepared as follows:- Solution 1

Aqueous suspension of spores in water with 0.01%

(vol) Triton X 100 wetting agent.

Solution 2

Spore suspension in 10% glycerol Solution 3

Spore suspension in water with 0.1% Triton B1956 emulsified in 5% canola oil.

Groups of eight plants, at 2-3 and 6-8 leaf stage, were each treated with one of the above solutions 1-3 by spraying the aqueous spore suspension onto the plant until run-off of the solution occurred. The treated plants were then covered with plastic bags, to maintain high humidity, for 20 hours. Temperature was maintained between 15 and 20°C. Control plants were sprayed with each of the three solutions, minus spores. No lesions or other effects were observed on the control plants.

Germinating spores on alisma plants treated with solution 3 developed appressoria at least 24 hours earlier than plants treated with solutions 1 or 2. Lesions developed on the younger leaves within 6-8 days.

Development of lesions was irregular typically up to 40 per leaf and lesions occurred 24-48 hours earlier in plants treated with solution 3 compared to plants treated with solutions 1 or 2, however, there was no significant difference between the final number of lesions per leaf between plants treated with the different solutions. Leaf death occurred 12-16 days after inoculation.

Example 2

7 Aqueous solutions containing 2.7 x 10 spores of Rhynchosporium alismatis per ml in 10% glycerol was prepared and inoculated onto seedlings by spraying. The seedlings were 3-4 strap leaf (i.e., 4 weeks old) at inoculation and had been grown submerged in 12cm of water. Water was drained off the seedlings 24 hours prior to inoculation. Flood water was reapplied to groups of 20 plants at either 24, 48 or 72 hours after inoculation. Control plants were sprayed with 10% glycerol only and flood water was reapplied at the above times.

The results obtained, as measured at 4 weeks after inoculation, are set forth in Table 3 below. An open ended growth scale was devised based on the number and type of leaves present (submerged or emergent). Emergent leaves were given a greater weighting than submerged leaves. The higher the number, the bigger the plant and the greater the competitive ability. TABLE 3

Hours Vigour score

(Time to reflooding) At inoculation 4 wks later

24 10 129

48 7 32 72 11 98

Control 12 403

All treated/inoculated plants were stunted compared to the control plants, however, it is clear that plants receiving 48 hours exposure to air prior to reflooding were affected the greatest by the treatment. In a field situation all the weed seedlings which are treated/ inoculated with the aqueous spore suspension would be outgrown by the rice and have minimal effect on the crop.

Field Trial

A field trial in the 1992/93 rice season at Yanco demonstrated the suppressive effects of the isolate

DAR67515 on the growth of A. lanceolatum. Six weeks after sowing into a rice bay, seedlings of

A. lanceolatum were sprayed with suspension of 2.23 x

7 10 spores/ml. Inoculum was applied late in the afternoon and flood water reapplied to the bays 48 hours later. The spores were suspended in water or an inoculum adjuvant comprising 0.01% wt of /3-Alanine (β-a) _r 10% wt of ^"glycerol (glycerol) or 0.1% wt hydroxy methyl cellulose

(HMC) . In each case the suspension contained 0.1% wt

Tween 20 surfactant. Plants were destructively harvested 53 days after inoculation. Scape number, scape height, leaf fresh and dry weights, root fresh and dry weights, total leaf fresh weight healthy leaf fresh weights, total leaf fresh weight healthy leaf fresh weight and leaf number per plot were all significantly reduced by application of the fungal spores (refer Table 4).

TABLE 4 Effect of four methods of suspending fungal spores on various growth parameters of A. lanceolatum

Inoculum adjuvant

Control Water β-a glycerol HMC sed* Significance nil vs treated

Scape number

1.04 0.22 0.44 0.22 0.06 0.2 P(0.001

Scape height (mm)

167 52 134 68 50 P(0.05

Scape fresh wt fmg) 2679 361 1214 567 22 685 P(0.001

Scape dry wt (mg)

409 41 130 89 111 P(0.001

Root fresh wt (mg)

60277 47175 50037 43002 45026 3338 P(0.001 Root dry wt (mg)

11419 8466 8869 7719 8875 865 P<0.001

Fresh wt healthy leaves (mg)

18496 6784 8473 6590 6428 1167 P(0.001

Dry wt healthy leaves (mg) 2481 908 1117 899 960 140 P(0.001

Total leaf fresh wt (mg)

37979 25886 32540 22726 23809 2381 P(0.001

Total leaf dry wt (mg)

5820 4281 5033 3818 3771 345 P(0.001 Total leaf number/plant

56.0 49.2 54.1 44.8 47.9 2.2 P<0.001

* Standard error of differences of the means. It can be seen that, in the treated plants, plant growth was reduced and maturity (measured as scape or infloresence stalk growth) was delayed. Quarantine Glasshouse Trial

Seedlings of Sagittaria guyanensis, S. pygmaea and Alisma canaliculatum were grown at the Plant Quarantine Station of the Biological and Chemical Research Institute at Rydalmere, New South Wales, Australia. The plants were sprayed to runoff with a spore suspension containing 5.21 x 10⁶ spores/ml of isolate DAR67513, 10% wt of glycerol and 0.1% wt of Tween 20 surfactant. Plants were maintained in closed containers for 18 hours to maintain a high humidity and reflooded 48 hours after inoculation. The results are shown in Table 5.

TABLE 5

Control Treated s.e.d Significance

Sagittaria guyanensis Plant height (mm) Leaf fresh weight (mg) per plot Leaf dry weight (mg) per plot Root fresh weight (mg) per plot Root dry weight (mg) per plot Sagittaria pygmaea Leaf dry weight

Alisma canaliculatum Leaf fresh weight (mg) per plot 8348 5818 920 P(0.05 Leaf dry weight (mg) per plot 372 244 45 P(0.05

Leaf number per plot 31 22 3 P<0.01 It can be seen that the leaf fresh and dry weights and leaf number per plot in A. canaliculatum were reduced. Leaf dry matter production in S. pygmaea was reduced (this is interesting as Korean research has shown that regeneration ability of the weed is reduced if leaves are cut during periods of active growth) . In S. guyanensis, plant height, leaf fresh and dry weights and root fresh and dry weights were all significantly reduced. In addition, there was high seedling mortality. The most beneficial use of the fungus will be inundative application of spores to the weeds at the seedling stage, causing death or stunting of the plant, reducing its competitive ability until it is overgrown by the rice crop. Glasshouse experiments show that combinations with very low doses of chemical herbicide may increase effectiveness of the disease. Current research elsewhere with mycoherbicides is highlighting the fact that plant death is not a necessary criterion for a successful agent:- reduction in competition for light/space/nutrients and decreased fecundity can be equally successful.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:-

1. A mycoherbicide for controlling weeds of the family Alismatacea , comprising fungal spores of Rhynchosporium alismatis , together with a suitable carrier therefor.

2. A mycoherbicide as claimed in claim 1 in which the fungal spores are present in the carrier as a substantially axenic suspension.

3. A mycoherbicide as claimed in claim 1 in which the spores are present in the mycoherbicide at a concentration of at least 8 x 10 spores/ml.

4. A mycoherbicide as claimed in claim 1 in which the fungal spores are derived from isolate N93/19114 deposited with the Australian Government Analytical Laboratory.

5. A mycoherbicide as claimed in claim 1 in which the carrier is a dilute surfactant solution in water optionally together with an inoculum adjuvant selected from the group comprising 3-alanine, glycerol and hydroxy methyl cellulose.

6. A method of controlling or eradicating weeds of the family Alismataceae, comprising applying to the weeds an effective amount of a mycoherbicide as claimed in any one of clams 1 to 5.

7. A method as claimed in claim 7 in which the weeds are selectively controlled in a crop, such as rice, a water channel or the like by the application of an effective amount of the mycoherbicide.

8. A method as claimed in claim 8 in which the weeds are selected from the group comprising Alisma lanceolatum, A. canaliculata_f A. plantago - aquatica, Sagittaria montevidensis, S. graminea, S. latifolia, S . pygmaea and Damasonium minus .

9. A method as claimed in claim 8 in which the mycoherbicide is a suspension of spores in a liquid and in which it is applied to the weeds until excipient or incipient runoff of the liquid suspension of spores occurs.