AU2015212307B2 - Mycoherbicidal composition for controlling Lantana camara weed - Google Patents

Abstract

The invention relates to describe host-specific extracellular phytotoxins produced by

Description

MYCOHERBICIDAL COMPOSITION FOR CONTROLLING LANTANA

CAMARA WEED

FIELD OF THE INVENTION

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 biocontrol activity. The present invention also discloses a molecular characterization of Fusarium sp that exhibit bio-control activity to Lantana camara.

BACKGROUND AND PRIOR ART OF THE INVENTION

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.

Present control methods are limited to physical removal and multiple application chemical herbicides. Plants larger than the fifth leaf stage are difficult to control with any of the commonly used herbicides. The fungus Fusarium sp has been used experimentally to control this weeds. A major constraint to commercial development of a plant pathogen and their metabolites as a biological herbicide is selectivity.

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There are many fungi which are pathogenic to weeds because they produce phytotoxins and, therefore, could be used as herbicides. The phytotoxins are 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 then they are considered to be definitive chemical probes in a study of disease susceptibility and physiological stress at the molecular level

During surveys of the fungal pathogens associated with L. camara were performed in Andhra Pradesh aimed at finding useful biological control agents. Among the fungi that were collected during such surveys was Fusarium Sp. Investigation of this fungus as a herbicide suggested production of an undefined toxin. This toxin may translocate between cells upon application of cell free extract upon host leaves causing foliar necrosis and blockage in stem parts that observed upon direct inoculation with Cell free culture filtrate.

One biorational approach to control this tenacious weed would consist of locating a host specific or selective pathogen of Lantana camara and employing either pathogen or its phytotoxic metabolites as control agents. During the search for black leaf blighted Lantana camara culminated in the discovery of an infected plant in Village Adavi Ramanpalam, Penuballi Mandal, Khammam District, Andhra Pradesh. The causal organism was identified as Fusarium on the basis of morphological and genetic study. The fungus was grown in liquid culture, and shown to produce a number of phytotoxin. To isolate the phytotoxin, 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.

OBJECTS OF THE INVENTION

In a first aspect, 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 In a further aspect 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.

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In yet a further aspect 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.

It is an object of the present invention to provide a novel herbicidal composition, a process for the preparation of the composition and a method using fungus or their extract, which has been developed for controlling Lantana and wherein said herbicide is a phytotoxin obtained from fungus Fusarium sp

Further herein described is a process for producing a novel herbicide composition effective against Lantana camara weeds said process 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.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

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, C18 Reverse phase Column elution profile of crude purified FO toxin in accordance with present invention;

Table 1: Fungi collected from Lantana camara

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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

Table 4: BLAST results of ITS-1, 5.8 S, and ITS-2 rDNA sequence data of AGLC#14

Table 5. Phytotoxicity of fermented broth of AGLC# 14 at different temperature

Table 6: Effect of AGLC 14 CFCF, crude filtrate and HPLC purified compound on the growth of lantana seedling.

Table 7: Effects of metabolites produced on excised leaves of Lantana

Table 8: Effects of metabolites of AGLC 14 on shoot cut of Lantana

Table 9: Host specificity testing

DETAILED DESCRIPTION OF THE INVENTION

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. Depending on the nature of the inhibitory substances in the cell free extracts, 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.

The 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. Additionally, 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 cellfree extracts in particular provides a significant advantage over existing biological control agents, in that the compositions and methods disclosed provide control of Lantana camara

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PCT/IN2015/000044 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.

Therefore, the present invention provides a herbicidal composition for controlling Lantana camara., 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. (AGBIO designated AGLC&14/NC1M 1372/MTCC 5975) but also indicated nucleotide variation of this fungus from other known genus of Nectria, Mariannaea, Volutella and Myrothecium as shown in (Figi). In addition AGLC#14 has surprising features in respect of herbicidal activity, in particular against Lantana camara sp.

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.

For applications, killed spores and/or cell free extracts of biocontrol 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.

For the phytotoxin extraction, cell free filtrate was extracted successively with hexane. Ethyl acetate. Chloroform and methanol. Phytotoxic compounds were detected both in the hexane fraction. Hexane fraction were collected and evaporated. This fraction named as crude filtrate. High Performance Liquid Chromatography (HPLC) was performed to partially purify the phytotoxic compound using methanol: water (0-100% gradient for 30 minutes) as solvent system in Cl8 reverse phase column. Collected fraction from HPLC is known as partially purified phytotoxin. A typical HPLC, Cl8 reverse phase column elution Profile of partially purified Toxin (FIG. 2). The separation of various peaks of AGLC#14 metabolites and its chemical characterization characterized by NMR and GC MS study and it will be added to the results, it has been protected by this patent.

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 100ml/l litre of

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PCT/IN2015/000044 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 2xl0⁸ 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. As a method for storing, 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 toxin activity in culture filtrates, as expressed by stem and foliar necrosis, was evident when intact seedlings, excised shoots or leaves were allowed to absorb the culture filtrate through their vascular system. In practice, it has been found that 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. However, 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.

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The 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. For compositions of the invention, 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. In a preferred embodiment, 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.

Other 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. When used, these additives should be used in an amount(s) which will not interfere with the growth, development or effectiveness of the biocontrol agent. Typically, 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. The 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 tw'o or more compositions applied sequentially, one of which

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PCT/IN2015/000044 contains live spores, and one of which contains killed spores and/or cell free extract to provide for protection at varying relative humidity. As will be apparent to one skilled in the art, 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. Further disclosed is 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 cunent growth; however, repeat applications may be necessary if regrowth of the plant occurs from resistant or below ground structures. Also 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.

The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1

Survey, Isolation and identification of host specific herbicidal strain:

The various regions of Andhra Pradesh has surveyed for collection of infected parts of Lantana leaf. Samples were transported to the laboratory in clean plastic bags and stored at 4° C until examined. Stored plant parts were scrubbed under running water to remove surface debris, dissected into small segments (approximately 1 * 1 cm), and surface-sterilized by sequential

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PCT/IN2015/000044 immersion in 96% ethanol for 30 seconds, 14% hypochlorite for 30 seconds and rinsed in sterile water for 1 min. Surface-sterilized segments from leaves were placed on Potato Dextrose plate supplemented with 0.5 mg streptomycin. The plates were incubated at 25°C for 5 to 7 days. The isolated and pure cultures were obtained. 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. During survey to Village Adavi Ramanpalam, Penuballi Mandal, Khammam District, Andhra Pradesh. Lantana leaves infected with pathogenic fungus Fusarium sp AGLC# 14 has shown maximum phytotoxicity toward this weed. Few genera of fungi were consistently isolated from the diseased Lantana, however, only AGLC14 fulfilled the Koch’s postulate and identified as Fusarium sp as per Singh 2007. The isolates were grown on the Potato dextrose agar medium and stock cultures of these were maintained at Laboratory and given Lab number AGLC 14. This strain has deposited in the NCIM, Pune. India and got an accession number NCIM 1372. A sample of an effective strain of Fusarium sp has been also deposited with IMTECH, Chandigarh under Budapest treaty, received accession number MTCC 5975 and identified as Fusarium moniliforme (Synonyms Gibberella fujikuroi).

Table 1: Fungi collected from Lantana camara

Pathogens	Symptoms	Lab code
Alternaria alternata	Leaf spot	AGLC04 AGLC05 AGLC 17 AGLC20
Colletotrichum sp.	Leaf spot	AGLC01 AGLC 16
Fusarium sp.	Leaf spot	AGLC07 AGLC08

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		AGLC10 AGLC14
Pestalotia sp	Leaf spot	AGLC02 AGLC03 AGLC06
Phoma sp.	Leaf spot	AGLC12 AGLC15 AGLC18
Curvularia sp	Leaf spot	AGLC11 AGLC13 AGLC19 AGLC21
Helminthsporium sp	Leaf spot	AGLC09

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 2xl0⁶ 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. In this isolate, 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

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PCT/IN2015/000044 intensity 6f 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#11, 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. The isolates disease range from 60-100% was selected for second trails.

Table 2: Pathogenicity test of fungi of Lantana camara

Pathogens	Accession number	Detached leaf bioassay
Alternaria alternata	AGLC04	2
	AGLC05	2
	AGLC 17	4
	AGLC20	4
Colletotrichum sp.	AGLC01	1
	AGLC 16	2
Fusarium sp.	AGLC07	2
	AGLC08	3
	AGLC 10	4
	AGLC14	5
Pestalotia	AGLC02	1
	AGLC03	1
	AGLC06	1
Phoma sp.	AGLC 12	1
	AGLC 15	4
	AGLC 18	1
Curvularia sp	AGLC 11	2
	AGLC 13	2
	AGLC 19	1
	AGLC21	2
Helminthsporium sp	AGLC09	2

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Abbreviation for detached leaf rating

0= no effect; 1= upto 20% leaf area necrotic; 2= Upto 40% leaf area necrotic; 3= upto 60% leaf area necrotic; 4= upto 80% leaf area necrotic; 5= Upto 100% leaf area necrotic

Shoot cut bioassay was carried out for assessment of herbicidal potential of the cell free culture filtrate. A gradient of 10% to 100% using sterile distilled water was prepared from stock cell free culture filtrate in test tubes. Potato broth uninoculated and sterilized distilled water served as control. Shoots were separated from healthy plants under water. These were surface sterilized using 90% alcohol and then placed in the gradients for bioassay. All treatments were carried out aseptically in duplicates for 48 hrs. The experiment was terminated at the end of 48 hrs and phytotoxic damage rating was done on five point scale where 0= No effect and 100% = complete collapse of the shoot (Walker and Templeton, 1978).

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.

Table 3 Screening of cell free crude filtrate of fungi for their phytotoxic activity against

Lantana catnara by cut shoot bioassay

Treatment	Strain no
Fusarium sp. AGLC 10	Fusarium sp. AGLC14	Phoma sp AGLC 15	Alternaria sp. AGLC 17	Alternaria sp. AGLC20
0 hi·	24 hr	36 hr	0 hr	24 hr	36 hr	0 hr	24 hr	36 hr	0 hr	24 hr	36 hr	0 hr	24 hr	36 hr
Control- Medium	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Control-	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

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Di st. water
10%	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
30%	0	0	2	0	2	3	0	0	0	0	1	1	0	1	1
50%	0	0	2	0	3	4	0	0	1	0	1	1	0	1	2
70%	0	2	2	0	4	4	0	1	1	0	1	1	0	1	3
90%	0	2	3	0	4	5	0	0	1	0	1	1	0	4	4
100%	0	4	4	0	4	5	0	1	2	0	1	1	0	3	4

Phytotoxic damage rating: —

0= no effect, 1 = slight chlorosis, 2= marked chlorosis/slight necrosis, 3= acute cholorsis/marked necrosis, 4= high necrosis/marked chlorosis, 5= acute necrosis and acute chlorosis leading to death.

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). The Symptom included from moderate chlorosis to marked chlorosis as well as necrosis, severe loss of water and drooping of leaves.

As par morphological analysis, indigenous strain AGLC#14(NCIM 1372/MTCC 5975) isolated from Lantana camara infected leaf is Fusarium sp. Macroscopic morphology as shown in Fig 3 may vary significantly on different media, and descriptions here are based upon growth on potato dextrose agar medium at 25°C. Rapid growth. Colonies woolly, frequently growing in blooms, initially white, becoming pink to red to brown centrally with the production of abundant chlamydoconidia. Reverse tan to brown to carmine red. As shown in Fig 4, the Hyphae are septate and hyaline. 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.

Example 2

Sequence homology and phylogenic analysis of AGLC#14

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The 18s TRNAsequence 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 Gibber ella 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.

Organism	Gene Bank Accession(s)	% identity (bp)
Fusarium moniliforme (Synonyms Gibberella fujikuroi)	AB237662	93%
Nectria mariannaeae	AB099509	92%
Mariannaea elegans var. punicea	ABH 1493	92%
Mariannaea camptospora	AB 112029	92%
Nectria mariannaeae	AB111492	92% .
Volutella ciliate	AJ301967	92%
Nectria cinnabarina	AB237663	92%
Myrothecium cinctum	AJ301996	91%
Myrothecium atroviride	AJ302002	91%
Myrothecium verrucaria	AJ302003	91%

Example 3

Production, extraction and characterization of herbicidal compound

Modified Liquid Minimal medium (MM) 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

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PCT/IN2015/000044 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 HC1. Culture filtrates (100 ml) were extracted three times into half the volume of n-hexane, chloroform, and methanol and ethyl acetate solvents separately. The organic phases were filtered through Whatman filter paper no.l and then evaporated to dryness at 30°C using a vacuum evaporator. Phytotoxic compounds were isolated by HPLC developed in the solvent system Methanol: Water. FIG. 2 depicts a reverse phase elution profile of partially purified Toxin.

To ascertain the mode of extraction of the phytotoxic moiety, it was extremely essential to determine the thermal nature of phytotoxin (s). For this, 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.

Table 5. Phytotoxicity of fermented broth of AGLC&14 at different temperature

Items	Phytotoxicity of cell free fermented broth at different temperature
40uC	50uC	60°C	100°C	120uC
12 H pt	24 hp t	48 hp t	12 hpt	24 hpt	48 hpt	12 hpt	24 hpt	48 hpt	12 hpt	24 hpt	48 hpt	12 hpt	24 hpt	48 hpt
AGLC#1 4	2	3	5	2	3	5	2	D	5	2	3	5	0	2	3
Control a	2	3	5	2	3	5	2	3	5	2	3	5	2	3	5
Control b	0	0	0	0	0	0	0	0	0	0	0	0	0	0 .	0

Phytotoxicity scale of 0 to 5 with 0 = no control, 5 = complete leaf necrosis HPT = hour post treatment

Ctrl A: Room temperature

Ctrl B: Broth medium.

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Example 4

Pathogenicity Tests of crude, cell-free filtrates and HPLC purified toxin on Lantana camara Seedling

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.

Table 6 : Effect of AGLC#14 on the growth of lantana seedling

Source	Toxin used (μΐ)	Phytotoxicity
Cell free culture filtrate	10.0	+++
Crude filtrate	5.0	+++
Partially purified	1.0	+++
Control	Distilled water	-

Example 5

Bioassay of crude, cell-free filtrates and HPLC purified toxin on Lantana camara detached leaves

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

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PCT/IN2015/000044 on the treated excised leaves were evaluated visually for damage for 5 days. The result of this test is depicted in Table 7.

Table 7: effects of metabolites produced by Fusarium on excised leaves of Lantana

Phytotoxin	Concentration (μΐ/ml)	Phytotoxicity
Cell free filtrate	100	+
Crude filtrate	10	+
Partially purified	0.2	+
Control	100	-

Ten leaves were used for each treatment. The phytotoxic damage was evaluated visually for 5 days. -= no phytotoxic effects, += phytotoxic effects.

Example 6:

Bioassay of crude, cell-free filtrates and HPLC purified toxin on Lantana camara shoot cut bioassay

Shoot cut Lantana bioassay was also used to test the, biological activities of crude, partially purified toxin and cell-and spore-free filtrates. Shoot were placed on test tube. The inocula of crude filtrates, cell-and spore-free filtrates, and the phytotoxin standards were filled on test tube to the shoot. Ten shoot were used for each treatment. Control leaves received distilled water. The test tubes containing shoot cut of Lantana were incubated under continuous or 12 h light. The phytotoxic effects on the treated shoot cut of Lantana were evaluated visually for damage for 3 days. Crude and cell-free filtrates and toxin caused damage to leaves, characterized by necrosis on leaves. The result of this test is depicted in Table 8.

Table 8: effects of AGLC&14 on Shoot cut of Lantana

Source	Concentration (μΐ/ml)	Phytotoxicity
Cell free filtrate	100	+
Crude filtrate	10	+
Partially purified	1	+
Control	NIL	- '
Ten leaves were usee	for each treatment. The phytotoxic damage was evaluated visually

for 10 days. -= no phytotoxic effects, += phytotoxic effects.

Example 7

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PCT/IN2015/000044

Host specificity of strain ,

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.

Table 9: Host specificity testing

Plants testes	Seedlings	Detached leaves
Chilly	-	NIL
Tomato	-	NIL
Brinjal	-	NIL
Parthenium	NIL	- .
l.antana	NIL	+++
Hyptis	NIL	-
Cassia tora	NIL	-

+++ = detached leaf necrosis - = no effect NIL = not tested

Although the foregoing description of the present invention has been shown and described with reference to particular embodiments and applications thereof, it has been presented for purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the particular embodiments and applications disclosed. It will be apparent to those having ordinary skill in the art that a number of changes, modifications, variations, or alterations to the invention as described herein may be made, in terms of dosage, dilution, application and the different forms of use, none of which depart from the spirit or scope of the present invention. The particular embodiments and applications were chosen and described to provide the best illustration of the principles of the invention and its practical application to thereby enable one of

WO 2015/114661

PCT/IN2015/000044 ordinary Skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such changes, modifications, variations, and alterations should therefore be seen as being within the scope of the present invention as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Claims (8)

1. CLAIMS:

   2015212307 22 Nov 2018

   1. A herbicide composition comprising an extract from a strain of a fungus Fusarium moniliforme f sp. laxmiae lab code AGLC&14 as deposited at NCIM and IMTECH under accession numbers NCIM 1372, and MTCC 5975, respectively, wherein the extract is selected from the group consisting of a spore inoculum, a conidial inoculum, a culture broth, an at least partially purified culture broth, a partially pure cell- and spore-free filtrate, a crude filtrate, and a crude suspension of the culture of Fusarium.
2. 2. The herbicide composition as claimed in claim 1, comprising one or more additives, such as conventional formulation additives, for example surfactants, solvents, carriers and diluents.
3. 3. A method for controlling Lantana camara weeds, said method comprising contacting the Lantana camara weeds with a herbicide composition according to claim 1 or claim 2, wherein said contacting preferably is achieved by applying the composition on the weed such as by spraying.
4. 4. A method as claimed in claim 3, wherein the extract is mixed with water, and suitable additives/formulation in a ratio of preferably 1:100.
5. 6. A strain of Fusarium moniliforme f sp. laxmiae being the strain of Fusarium moniliforme AGLC&14 as deposited at NCIM and IMTECH under accession numbers NCIM 1372, and MTCC 5975, respectively.
6. 7. A herbicidal extract obtained by a method comprising hexane, ethyl acetate, chloroform and/or methanol extraction of Cell Free Culture Filtrate of Fusarium moniliforme f sp. laxmiae being the strain of Fusarium moniliforme lab code AGLC&14 as deposited at NCIM and IMTECH under accession numbers NCIM 1372, and MTCC 5975, respectively.
7. 8. Use of a Fusarium moniliforme f sp. laxmiae, being the Fusarium moniliforme strain AGLC&14 as deposited at NCIM and IMTECH under accession numbers NCIM 1372, and MTCC 5975, respectively, for producing an extract effective for controlling growth of Lantana camara, wherein the extract is selected from the group consisting of a hyphal inoculum, a spore inoculum, a conidial inoculum, a culture broth, and an at least partially purified culture broth.
8. 9. Use according to claim 8, wherein the extract is obtained from at least one of the solvents selected from the group consisting of hexane, ethyl acetate, chloroform and methanol, by extraction of Cell Free Culture Filtrate of the Fusarium sp.