WO2015114661A1 - Mycoherbicidal composition for controlling lantana camara weed - Google Patents
Mycoherbicidal composition for controlling lantana camara weed Download PDFInfo
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- WO2015114661A1 WO2015114661A1 PCT/IN2015/000044 IN2015000044W WO2015114661A1 WO 2015114661 A1 WO2015114661 A1 WO 2015114661A1 IN 2015000044 W IN2015000044 W IN 2015000044W WO 2015114661 A1 WO2015114661 A1 WO 2015114661A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
Definitions
- the present invention provides a novel herbicidal isolate of Fusarium sp AGLC #14 or partially pure or cell-and spore-free filtrate or crude filtrate or a crude suspension obtained therefrom, useful for the control of Lantana camara.
- the present invention also discloses herbicidal compositions comprising fungal isolates formulated in a growth medium for maintaining the viability of the product when the biological control composition is applied to weed.
- the present invention also discloses methods of screening fungal isolates to determine if they exhibit bio- control activity.
- the present invention also discloses a molecular characterization of Fusarium sp that exhibit bio-control activity to Lantana camara.
- Lantana camara is a native of tropical America, and was introduced to India as an ornamental to be planted in gardens and hedges. Since then, the species has spread rapidly into both farm and forest lands, and is one of the most widespread, terrestrial invasive species in India today. It is considered as one of the world's 100 most invasive species, and among the world's 10 worst weeds. Lantana grows on all types of well-drained soils and in a wide rainfall range (from seasonal dry forests to rainforest) but is also very drought-resistant. It rarely invades undisturbed, closed-canopy forest but rapidly colonizes gaps, edges and disturbed or logged habitats. It produces large numbers of seeds that are dispersed by birds and the seeds germinate rapidly and easily.
- Lantana is a very efficient competitor against native species under conditions of high light, soil moisture and soil nutrients. It can become the dominant understorey species in infested areas, blocking natural succession processes, and reducing biodiversity. It may be threatening the wildlife habitats in forests, and thereby threatening important wildlife populations.
- the applicants cultured the fungus FO in a defined medium for 1 to 7 days and the cell free filtrate was analyzed for phytotoxic activity using shoot cut and seed bioassay of Lantana camara as post-emergent.
- the cell free filtrate exhibited phytotoxicity.
- the present invention provides a herbicidal composition suitable for controlling Lantana camara the composition comprising a herbicidal agent from a Fusarium strain.
- Fusarium sp sp. is on deposit in the patent collection of the IMTECH under the terms of the Budapest Treaty and has been assigned accession number MTCC 5975. It is also deposited in NCIM under accession number 1372
- the present invention relates to a method for controlling growth of plants from Lantana camara sp., comprising contacting the Lantana plants with the herbicide composition according to the invention.
- the invention relates to use of an Fusarium strain for producing a herbicidal agent effective for controlling growth of Lantana plants.
- the strain used preferably is a strain having the characterizing features of Fusarium strain AGLC#14 as deposited at IMTECH under accession number MTCC 5975 and finally identified as Fusarium moniliforme.
- Another object of this invention is to provide a novel herbicide effective against herbs Lantana comprising a phytotoxically effective amount of the toxin produced by Fusarium sp together with an additive or a carrier compatible with the said toxin and soil environment.
- a process for producing a novel herbicide composition effective against Lantana camara weeds comprising phytotoxin from Fusarium sp., said process comprising: (a) Culturing the Fusarium obtained from a infect leaves of Lantana at time of survey; in a PDA culture medium for about 7 days, (b) Separating the phytotoxins by Cell free culture filtrate from the culture medium by Solvent extraction methods, and; (c) Dispersing the CFCF with suitable carriers or additives at an application rate of active agent ranging from about 100 ml/ 1 litre of water to obtain herbicidal composition.
- Fig. 1 illustrates phylogenetic relationship of fungi to selected species from the similar genera based on 18s rRNA genes in accordance with present invention
- Fig. 2 illustrates a typical HPLC, C 18 Reverse phase Column elution profile of crude purified FO toxin in accordance with present invention
- Table 1 Fungi collected from Lantana camara
- Table 2 Pathogenicity test of fungi of Lantana camara
- Table 3 Screening of cell free crude filtrate of fungi for their phytotoxic activity against Lantana camara by cut shoot bioassay
- the present invention relates to weed control compositions comprising isolate's spores, cell free culture filtrate, crude metabolites and pure purified phytotoxin effective for the control of Lantana weeds which is a troublesome weed in the cultivation of economic crops and in forest.
- the present invention is directed to methods for the control of Lantana camara by the application of fungal products, particularly those derived from of fungi.
- Cell-free extracts of fungi can be prepared by filtering culture medium in which fungi has been grown to remove any cells and is optionally sterilized by procedures known to those of skill in the art.
- the extracts are stable to 15 minutes at 100°C temperatures.
- the inhibitory agents may be purified using means well-known to those skilled in the art, including one or more of column, thin layer, gel and capillary chromatography, high pressure liquid chromatography, mass spectroscopy, or a combination of high pressure liquid chromatography and mass spectroscopy.
- the cell free extracts are formulated in an agriculturally compatible medium for plant application.
- compositions used include killed spores and a cell-free extract of fungal culture.
- the present high density spore preparations are especially effective in arresting growth of Lantana camara.
- the killed spore preparations and cell-free extracts have a long shelf life and are more stable than compositions comprising living organisms.
- the use of killed spores and/or cell- free extracts in particular provides a significant advantage over existing biological control agents, in that the compositions and methods disclosed provide control of Lantana camara independent of humidity level; relative humidity in the field may fall well below the level necessary for germination and subsequent growth of weed pathogenic fungi.
- the present invention provides a herbicidal composition for controlling Lantcma c m ra., the composition comprising a herbicidal agent or phytotoxin from an Fusarium strain.
- the Fusarium strain preferably is a strain having the characterizing features of Fusarium sp strain AGLC#14 as deposited at 1MTECH under accession number. MTCC 5975. A characterizing feature of this strain, is its 18s rRNAsequence. The 16srRNA sequence analysis showed 93% nucleotide similarity of the Fungi to Fusarium sp sp.
- AGLC#14 NCIM 1372/MTCC 5975
- Fig 1 Fig 1
- AGLC#14 has surprising features in respect of herbicidal activity, in particular against Lantcma camara sp.
- biocontrol agent For agricultural applications particularly field use. large scale production of biocontrol agent is necessary. Large scale production of biocontrol agent is carried out by growing the spores on suitable medium.
- killed spores and/or cell free extracts of biocontfol agent have proven to be effective in preventing growth of weeds.
- the killed spores can be prepared by any method well known to those skilled in the art. including heating by boiling, autoclaving, steaming and the like.
- the herbicidal compositions of the invention are prepared by dispersing the cultures in suitable medium at an application rate of active agent, preferably ranging from about lOOml/l litre of water. About 1.0 to 1.5 litre of formulated cell free broth per acre of the target area to be treated, depending on the severity of the infestation, the condition of the target area and applied by method of spraying and the like.
- the herbicide of the invention may be formulated by suspending in water spores obtained by mass-culturing, but the formulation method is not limited to this one. In this method, the spore concentration is appropriately 2xl0 8 spores/ml but not limited to this range. In suspending spores in water, adjuvants such as a surfactant and a spreader may be added.
- the Fusarium sp strain which is the major agent may be a fresh strain immediately after fermentation. Alternatively, a once stored strain may be used after renaturing with water or the like.
- well known methods for storing strain such as ultra low temperature storing (-10 DEG C), vacuum lyophilization or the like may be used.
- Water is a suitable medium for dispersing toxin-containing cultures. It is suitable to use formulations of toxin from crude fungal inocula or fractions thereof, such as cell-free filtrates, thereby obviating the need to isolate the pure compound.
- the cell free filtrate, crude filtrate as well as the partially purified toxin is effective in controlling weeds. Therefore, the cell free culture filtrate may be used as such, if desired, in the herbicidal composition, thereby obviating the need for any purification steps.
- formulations of culture broth are definitely the most preferred and suitable.
- the said composition may comprise a number of additives, such as conventional formulation additives for examples surfactants, solvents, carriers and diluents.
- Effective microbial weed control compositions and microbial origin weed control compositions can be produced by mass-culturing Fusarium sp. and efficiently obtaining spores, both under aseptic conditions. When applied to the weed, these weed control compositions possess selective herbicidal activities against Lantana only and show substantially no pathogenicity against economic crops and other weeds. They therefore have highly-selective herbicidal activities, and are free of potential problem of environmental contamination and thus can be used safely.
- compositions and methods of the invention include the provision of application of cell free culture filtrate or their metabolites to produce a novel herbicide
- herbicide compositions may be prepared as a liquid formulation by suspending the broth, cell free broth, crude or purified metabolites in an agriculturally acceptable carrier for application to the weed or the location where it is growing.
- any agriculturally acceptable carrier can be used whether it is liquid or solid as long as it can be employed in agricultural or horticultural formulations and is preferably biologically inert.
- Exemplary agriculturally acceptable liquid carriers include, but are not limited to, water, surfactants, vegetable oils, and mineral oils.
- the agriculturally acceptable carrier for a liquid formulation is water, and the herbicide has a cell free broth.
- the herbicide compositions can also be prepared as granular formulations, flowable formulations, or wettable powder formulations by mixing with an agriculturally acceptable carrier, which is then applied to weed.
- Suitable agriculturally acceptable solid carriers include mineral powders, such as clay, talc, bentonite, calcium carbonate, diatomaceous earth and white carbon; vegetable flours such as soybean flour and starch, and some polymers such as polyvinyl alcohol and polyalkylene glycol.
- additives known to the art can be added to the agricultural formulation, including additives which promote spreading of the compositions of the present invention, additives such as surfactants which render the formulations substantive so as to increase the ability of the formulations to stick to parts of the part that are contacted with the formulation, nutrients for the fungal cells when live spores are used, and mixtures of the aforementioned additives.
- additives such as surfactants which render the formulations substantive so as to increase the ability of the formulations to stick to parts of the part that are contacted with the formulation, nutrients for the fungal cells when live spores are used, and mixtures of the aforementioned additives.
- these additives should be used in an amount(s) which will not interfere with the growth, development or effectiveness of the biocontrol agent.
- preparation of suitable compositions requires only mixing of the biocontrol agent with the additives. Typical preparation includes: adding together the fungal organism(s), preservative and powdered ingredient; and then mixing and/or grinding the constituents together.
- compositions of the present invention will have excellent storage properties, will not require refrigeration, will not typically encounter contamination problems, and will remain effective in typical agricultural storage environments for relatively long periods of time.
- the application may utilize a single composition comprising the bioagent preparation or two or more compositions applied sequentially, one of which contains live spores, and one of which contains killed spores and/or cell free extract to provide for protection at varying relative humidity.
- effective concentrations of the bioagent preparations may vary depending upon such factors as: (1 ) the physiological condition of the plant (e.g. the stage of the growing season); (2) temperature and humidity; and (3) region or locale. The optimal concentration for any application may be determined by the skilled artisan through known testing procedures.
- the composition comprises a herbicidal agent is selected from a spore inoculum, preferably a conidial inoculum, culture broth, preferably an at least partially purified culture broth, partially pure, a cell-and spore-free filtrate, a crude filtrate, or a crude suspension of the culture of Fusarium or a secondary metabolite.
- a method of controlling weeds includes applying the herbicide containing cell free broth or their purified metabolites.
- the herbicide can be applied by spraying containing solution of fungal metabolites at weed in an amount sufficient to coat the leaves of the weed.
- One application may be sufficient to reduce current growth; however, repeat applications may be necessary if regrowth of the plant occurs from resistant or below ground structures.
- the method of the present invention may be used in addition to or in conjunction with other control measures.
- Pathogens useful in the practice of this invention were selected by conducting herbicidal activity tests on weeds and crops with respect to strains isolated from naturally-infected Lantana. As a result of a morphological identification of the obtained strains with the activity of the controlling Lantana camara, they were found to be classified as Fusarium sp [AGLC 14]. These depositions were converted to depositions under the requirements of the Budapest Treaty and got a accession number MTCC 5975 The culture is also deposited in the NCIM, Pune, India and got an accession number NCIM 1372.
- Identification of the different fungal genera was based on morphological characteristics of each growing microbial colony. Fungi having distinctive characteristics of saprophytes (i.e. rapid growth and sporulation on PDA plates were excluded from further consideration after their initial isolation. The retained necrotrophic fungal isolates were identified. The fungal isolates were stored at Lab. The infecting fungi (21 isolates of fungi) were identified as belonging to 7 known genera (Table 1 ) on the basis of their morphological characteristics and the arrangement and structure of their conidia.
- Fungal inoculums were prepared by fungal pathogens recover during survey. They were extensively tested for their herbicidal potential. The conidia or mycelial fragments were harvested by flooding presterilized PDA petri dishes having pure culture with sterile distilled water. The spore suspension including 2xl0 6 spores/ml was adjusted with haemocytometer. The potential of these isolates were assessed using detached leaf bioassay method (Winder and Watson, 1994). Excised third leaf from preflowering stage of the weed from fields was placed in a moist chamaber. The inoculums were applied to them in an even coat with bristles of an artist #6 brush. Controls sets were and sprayed with sterile distilled water and surfactant only. This was done within 15 minutes of detachment of leaves. Inoculated detached leaves were incubated for 4 days at room temperature. Detached leaves were rated for disease severity 4 days after treatment on a 5 point sale.
- Results presented in Table 2 reveals that seven fungal species were identified from disease leaves and stem parts of Lantana camara and several isolates incite severe infection and caused considerable damage of the weed in detached leaf bioassay.
- Fusarium sp (AGLC#14) incite maximum infection and colonized more rapidly than others.
- infection started from the base and margins of leaves, which turns dark brown to black after 48 hr of incubation. Abundant sporulation and mycelial growth of the pathogen was appeared on the leaf surface. The intensity of the disease also ranged 80-90% in case of Fusarium sp (AGLC#14).
- the LAD was 30-40% in case of AGLC#04, AGLC#05, AGLC#07, AGLC#09, AGLC#1 1 , AGLC#13 and AGLC#16. Rest of the isolates viz., AGLC#01 , AGLC#02, AGLC#03, AGLC#06, AGLC#12, AGLC#18 and AGLC# 19 caused 10-20% LAD.
- LAD was 60-80% in case of AGLC#08, AGLC#10, AGLC#15. AGLC#17 and AGLC# 20.
- Fusarium sp A thorough screening of Cell free culture filtrate of fungi for their phytotoxic activity led to selection of potential strains viz. Fusarium sp (AGLC#14). Symptoms of phytotoxic damage were marked chlorosis, marginal necrosis, and loss of leaf turgidity (crumbling). Fusarium sp (AGLC# 14) showed marked phytotoxic damage symptoms at 50% to 90% concentration of crude culture filtrate at 24 hpt and 36 hpt (Table 3). The Symptom included from moderate chlorosis to marked chlorosis as well as necrosis, severe loss of water and drooping of leaves. Fusarium sp (AGLC#14) have been selected for further studies.
- Conidiophores are simple or branched monophialides and polyphialides (phialides with more than one opening not delimited by a septum). Microconidia are abundant, spindle-shaped, 0-3 septate (never globose). Macroconidia is rare, 3-5 septate, sickle-shaped.
- the 18s 'rRNAsequence analysis showed 93% nucleotide similarity of the Fungi to Fusarium sp but also indicated nucleotide variation of this fungus from other known genus of Nectaria, Mariannaea. Volutella, and Myrothecium (Table 4).
- the invention broadly pertains to a novel isolate of Fusarium sp that exhibits host specific toward Lantana camara weeds.
- the 18s rRNAsequence analysis showed 93% nucleotide similarity of the Fungi to Fusarium moniliforme (Synonyms Gibberella fujikuroi, perfect stage of Fusarium moniliforme species) but also indicated nucleotide variation of this fungus from other known genus of Fusarium (Fig 1).
- the fungal isolates AGLC#14 was characterized based on partial DNA sequence of the internal transcribed spacer regions of the nuclear ribosomal RNA gene.
- the ITS sequence was obtained using the deposited database of ITS. Both isolates ITS sequence obtained was not 100% identical to any ITS sequence deposited in Gene Bank.
- Table 4 BLAST results of ITS-1 , 5.8 S, and ITS-2 rDNA sequence data of AGLC#14.
- Modified Liquid Minimal medium was prepared. The medium was distributed in 100 ml aliquots in one liter flask and after autoclaving at 121 °C for 15 min and cooling, inoculated with two 2mm disc of 6-8 days, preferably 7 days, old culture of AGLC 14 isolate. Flasks were incubated at 25°C in dark without shaking. Culture filtrates were harvested after 14 days by filtering through muslin cloth and obtained filtrate filtered through Whatman filter paper#01. PH of the culture filtrates was adjusted to 3.0 with 2 M HCl. Culture filtrates (100 ml) were extracted three times into half the volume of n-hexane, chloroform, and methanol and ethyl acetate solvents separately.
- MM Modified Liquid Minimal medium
- FIG. 2 depicts a reverse phase elution profile of partially purified Toxin.
- CFCF of AGLC#14 was subjected to different temperature treatment viz. 40, 50, 60, 100 and 120°C (autoclaved). Each treatment was carried out for 15 mins. The phytotoxic activity of each treatment was assessed using the shoot cut bioassay. Each treatment was carried out in triplicate and CFCF at room temperature served as control and uninoculated medium served as second control. Results showed in table 5 that the phytotoxicity of fermented broth of AGLC#14 did not change and was stable at 40, 50, 60 100°C and 120°C on the leaf of weeds.
- Lantana seedling was planted in pot. The plants were watered as needed. The photoperiod was 14 h. The fungal inoculum was applied using a sprayer to run-off. Control groups received a filtrate of autoclaved distilled water. Lantana seedlings were used in these experiments. Following inoculation, plants were incubated on greenhouse benches under conditions as described above. Three replicates of 10 plants each were used for each treatment. The experiment was repeated three times. Symptom development was monitored daily. Results were observed at the beginning and the end of the experiments. All treated plant showed wilting and complete necrosis. The results are shown in Table 6. In seedling, the damage resulting from the crude, cell-free filtrates and partially purified toxin were identical, including visibility of necrosis of the leaves of the weeds. Sterilized distilled water was used as controls for the method.
- Detached Lantana leaves were used to test the, biological activities of cell free filtrates, crude filtrate and partially purified toxin. Detached leaves were placed on moistened filter paper inside 9-cm diameter sterile Petri plates. The inocula of crude filtrates, cell-and spore-free filtrates, and the partially purified phytotoxin were applied to the leaves with micropipets. Ten leaves were used for each treatment. Control leaves received distilled water. The plates were sealed with parafilm and incubated at Room temperature under 12 h light condition. The phytotoxic effects on the treated excised leaves were evaluated visually for damage for 5 days. The result of this test is depicted in Table 7.
- Three weeds and three crops were used for testing host specificity of strain AGLC#14.
- the crops plants are ranged in age from seven to ten days old at the time of spraying. Seeds used in these experiments were or collected locally. Seeds of each crops viz. chilly, brinjal and tomato were planted. The experiment was confirmed by repeating twice.
- One concentration of cell free culture broth 1% was prepared by adding 10 ml in 1 litre distilled water.
- a sprayer was used to apply the toxin solutions until run off. Plants were kept in the greenhouse under the same conditions as described in earlier.
- the three weeds viz. Parthenium, Lantana, Hyptis and Cassia tora detached leaves were treated as detached leave bioassay method. Symptoms were observed daily until the end (two weeks) of the experiment and included chlorosis, necrosis, stunting and mortality. The results are shown in Table 9.
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AU2015212307A AU2015212307B2 (en) | 2014-01-27 | 2015-01-23 | Mycoherbicidal composition for controlling Lantana camara weed |
ZA2016/05951A ZA201605951B (en) | 2014-01-27 | 2016-08-26 | Mycoherbicidal composition for controlling lantana camara weed |
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