AU2018267591A1 - An Herbicidal Composition for Controlling Parthenium Weed and Strain Thereof - Google Patents

Abstract

The present invention relates to the development of novel mycoherbicide for controlling Parthenium hysterophorus. The present invention also discloses fungal isolates of Altemaria sp AGPH#04 use spores, as a cell-and spore-free filtrate, a crude filtrate, or a crude suspension of the culture and optionally along with other additives. The fungal strain was observed to be particularly virulent against Parthenium. The fungal strain was deposited in NCIM as private culture collection and assigned the number NCIM 13 71. The strain was also deposit in the patent collection of the IMTECH under the terms of the Budapest Treaty and has been assigned accession number MTCC 5973. The sequence homology and phylogenic analysis of strain showed 98% nucleotide similarity with other similar genus isolate. The present invention also discloses whole genome study for identification of novel probe and primer pair sequence for use in detecting isolates that exhibit bio-control activity. The present invention also discloses process of production and characterization of naturally occurring host specific phytotoxic compound that is excreted by the Alternaria sp. AG-PH04.

Description

FIELD OF THE INVENTION

The present invention provides a novel herbicidal isolate of Alternaria sp. or celland spore-free filtrate or crude filtrate or a crude suspension or partially pure obtained therefrom, useful for the control of Parthenium weeds. 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 whole genome study for detecting Alternaria sp. isolates that exhibit biocontrol activity to Parthenium weeds.

BACKGROUND AND PRIOR ART OF THE II

Parthenium is regarded as one of the worst weeds because of its invasiveness, potential for spread, and economic and environmental impacts, and it is noxious because it is highly adaptable to almost all type of environmental conditions, can invade all types of land, also causes high losses in the yield of field crops and direct contact with plant or plant parts for long time causes dermatitis sometimes it may lead to death of person, fever and asthma.

Parthenium is hazardous to human health. Health impacts on humans include contact dermatitis, skin irritation, nausea, giddiness and respiratory problems like bronchitis and asthma, eye irritation, and sinusitis (hay fever). Sesquiterpene lactones specially Parthenin found in almost all the plant parts are causing these hazards. Parthenium causes acute toxicity in cattle and milk becomes bitter tasting due to the presence of parthenin, which is also hepatotoxic. If it is present in animal diet then causes dermatitis with pronounced skin lesions and a significant amount (10-50%) of P. hysterophorus in the diet can kill cattle and buffalo. In India, P. hysterophorus causes a yield decline of up to 40% in agricultural crops. Due to the invasive capacity and allopathic effects of allelochemicals, phenolics and, sesquiterpene lactones, mainly parthenin, it inhibits the

2018267591 20 Nov 2018 germination and growth of plants including pasture grasses, cereals, vegetables and other plant species. Thus, displacing native plant species and transforming grasslands, wasteland pastures, open fields to monocultural shrub lands. In agricultural fields, it depletes nutrients from and releases toxic substances into the soil. Thus, affects soil quality and reduces yield of crops. Parthenium pollen can also inhibit fruit set in beans, eggplant, peppers, tomatoes, and other plants. Because their extremely large no. in the field area inhibits the pollens of crop plant to disperse and pollinate. Contamination of crop plant seeds with Parthenium seeds restricts their sale and export. By acting as host for insects such as cotton mealybug and disease-causing organisms such as tomato leaf curl virus, it promotes spreading of these diseases in the field. It is commonly called as congress grass, carrot weed, white top etc. Some of the local names by which Parthenium is known are chatak chandani, broom brush, gajari and safedtopi. It is among the top ten worst weeds of the world and has been listed in the global invasive species database. It is widely occurring and occupied almost all the parts of world such as in Asia (Bangladesh, India, Israel, Pakistan, Nepal, southern China, Sri Lanka, Taiwan and Vietnam), Africa (Ethiopia, Kenya, Madagascar, Mozambique, South Africa, Somalia, Swaziland and Zimbabwe), Australia and the Pacific (New Caledonia, Papua New Guinea, Seychelles and Vanuatu).

It is native of Northeast Mexico, and introduced in India along with wheat grains under the PL 480 scheme and then spread in almost all the states in India and gets established in almost all the parts such as wasteland, roadsides, forests, railway tracks, and crop fields. In 2005, it was estimated that it infested over five million acres of the Indian subcontinent. It is an invasive alien species; these are such species whose introduction or spread threatens the environment, economy or society including human health. Its high germination ability throughout the year, an enormous seed bank, rapid spread and colonization and extreme adaptability in a wide range of habitats make it capable of spread vigorously. It produces up to 25,000 seeds per plant arid dispersal of this large seed bank in the soil account for its vigorous spreading. Its deep penetrating roots, erect shoot system and suppression of growth of other native species with its allopathic effect are major supports that help in its rapid establishment in any type of environment.

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Furthermore, it is a serious threat to native fauna and flora and can causes severe reductions in biodiversity. The spread of weed can be controlled by the use of an integrated range of methods- mechanical, cultural, chemical, and biological. Manual uprooting of Parthenium before flowering and seed setting is the most effective method. This is easily done when the soil is wet. Uprooting the weed after seed setting have two main drawbacks i.e. it will increase the area of infestation and pulling a plant in flower will aid in the dispersal of pollen grains, resulting in allergic reactions. Ploughing the weed in before the plants reach the flowering stage and establishing pastures or other plants may be effective. However, the manual removal is usually neither very effective nor economical, because of the rapid regrowth requiring repeated removals for seasonlong control. Several cultural practices also used such as preventing introduction of Parthenium seeds by keeping clean the equipments, livestock, animal feed, people, and vehicles, preventing physical spread of the seeds by cultivators, shoes, tires, machinery.

Parthenium weed can be suppressed by growing competitive crops (fodder sorghum sunflower and maize) or self-perpetuating competitive plant species like Cassia sericea, C. tora, Tagetuserecta (marigold), and Abutilon indicum, Croton bonplandianus and C. sparsiflorus, Cassia auriculata, in non-crop areas which will cofnpete with the weed and reduce its population. In certain parts of India, crop rotation using marigold (Tagetes spp.) during rainy season, instead of the usual crop, is found effective in reducing parthenium infestation in cultivated areas. Burning can kill Parthenium weed above ground plant parts and seed near the soil surface, but buried seeds may survive. The use of synthetic herbicides such as alachlor, paraquat, simazine, 2, 4-D, 2, 4, 5-T, glyphosate, Atrazine, and Metribuzin is effective. But the timing of chemical control is critical. The plants should be treated before flowering and seed setting and when other plants, especially grass, are actively growing and can recolonize the infested area. In open wasteland, noncropped areas and along railway tracks and roadsides, the spraying of a solution of common salt (Sodium chloride) at 15- 20% concentration has been found effective. However, excessive use of herbicides causes threat to environment and also these does not affect the underground plant parts of weed, like root stumps, stolons or

2018267591 20 Nov 2018 suckers of the weeds, Seeds in the soil also germinate and thus' the plants soon emerge arid colonize. Excessive dependence on the herbicides has produced more resistant weeds.

Herbicide resistance development in chickweed substantially limits herbicide choices for its control in most crops and other non-agricultural lands. Thus, there exists a need in the art for improved and safer compositions and methods for controlling weed species to replace the synthetic herbicidal chemicals currently used for this purpose.

This situation provides an opportunity to develop bioherbicide or microbial herbicides or “mycoherbicide” for Parthenium control. Some microorganisms produce substances that are toxic to plants. One strategy for developing less environmentally harmful herbicides is to identify and isolate phytotoxins produced by microorganisms. If such toxins can be used as herbicides, then these naturally-occurring agents may have fewer deleterious environmental effects than the synthetic chemicals currently used for weed control. Effort has been directed at identifying and isolating phytotoxins made by microorganisms for possible use as herbicides. Such efforts have usually involved attempts to identify and isolate phytotoxins from culture media in which bacteria or fungi have grown. ¹

The isolation and identification of herbicidal compounds made by microorganisms faces numerous obstacles. The process of identifying, purifying, and isolating such compounds is lengthy and expensive. Media in which microorganisms have grown (conditioned media) are highly complex mixtures and the active compounds with herbicidal activity may be present iri extremely low concentrations. If the active compounds are labile, or if herbicidal actions are due to synergistic interactions of multiple components, then the activity may be lost during purification. Even where an active compound is identified, the production of commercially useful quantities of such compounds by industrial fermentation and purification or by chemical synthesis may require considerable additional effort.

Parthenium management through using natural herbicides from fungi is a new kind of bioherbicide as an alternative to chemical herbicide. Mycoherbicides can be divided into

2018267591 20 Nov 2018 fungus preparation herbicide and fungus derived herbicide by virtue of the effective components from the pathogen itself or its phytotoxin. In last 10 years, some phytotoxin screened from pathogenic weeds showed potential herbicidal activity. Some experts suggest these phytotoxin be developed as new kind of Bioherbicide as an alternative chemical herbicide. Phytotoxin used for mycoherbicide can be fungal derived product. The pathogen which produces phytotoxins as a microbial herbicide must fit certain requirements: (i) Be reproduced by biological techniques, (ii) grow fast after spraying or be capable of killing weeds within definite time, (iii) suit industrial production and (iv) suitable for packaging, transport and use. Because the fungal toxin used in mycoherbicides usually occur naturally in the areas where they are utilized, they tend to be less harmful to the environment than chemical herbicides. The fungal toxin are often more selective in their mode of action so the risk of damage to other plants is reduced. Mycoherbicides are, as a rule, less toxic to people and animals than chemical herbicides. The applicants in the course of their studies on mycoherbicide development found that Parthenium weed is susceptible to attack by micro-organisms and most of the pathogens attacking the plant have been identified. The applicants have also found that a fungus Alternaria sp. which is responsible for the leaf disease in weed, releases certain toxins which exhibit phytotoxicity against Parthenium. The first whole genome sequence of Alternaria sp AGPH#04 is also presented. The size of complete whole genome was determined to be 3882817 bp, having a total of 273 contigs and GC% is 52.9.

OBJECTS OF THE INVENTION

In a first aspect, the present invention provides a herbicidal composition suitable for controlling Parthenium sp., the composition comprising a herbicidal agent from an Alternaria strain. Alternaria sp AGPH04 was deposited in the patent collection of the IMTECH under the terms of the Budapest Treaty and has been assigned accession number MTCC 5973 and identified as Alternaria alternata. It is also deposited in NCIM under accession number 1371. In a further aspect the present invention relates to a method for controlling growth of Parthenium weed comprising contacting the Parthenium 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 Alternaria sp AGPH 04 for producing herbicidal agent effective for controlling growth of Parthenium plants. The present invention also discloses process of production and characterization of naturally occurring host specific phytotoxic compound that is excreted by the Alternaria sp. AGPH04.

In yet a further aspect the invention also relates to study of sequence homology and phylogenic analysis of strain showed 98% nucleotide similarity with other similar genus isolate. The present invention also relates to whole genome study for identification of novel probe and primer pair sequence for use in detecting isolates that exhibit bio-control activity. This is first report of whole genome study of mycoherbicidal strains Alternaria sp AGPH04 against Parthenium weed.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Fig 1 illustrates the macroscopic morphology of Alternaria in accordance with present invention;

Fig 2 illustrates the microscopic morphology of Alternaria in accordance with present invention;

Fig. 3 illustrates phylogenetic relationship of Alternaria sp. AGPHA04 to selected species from the genera Alternaria based on 18S rRNA genes in accordance with present invention; '

Fig. 4: Schematic diagram for extraction of phyto toxic compound from AGPH04

Fig. 5 illustrates typical HPLC, Cl8 reverse phase column elution Profile of crude puri fied Toxin in accordance with certain embodiments of present invention;

Fig 6: The crude brown metabolite (oily residue) of AGPH04

Fig 7 and Fig 8 illustrate base quality score distribution, left (Rl) and right (R2) end of the paired end read for the fungal sample AGPH04in accordance with present invention;

Fig 9 and Fig 10 illustrate base composition distribution of left and right end of the paired end read sequence for the fungal sample AGPH04in accordance with present invention;

Fig 11 and Fig 12 illustrate The GC distribution of left and right end of the paired end read sequence for the fungal sample AGPH04in accordance with present invention;

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Fig 13 illustrates BLASTX E Value distribution for the fungal sample AGPH04in accordance with present invention;

Fig 14 illustrates BLASTX similarity score distribution for the fungal sample AGPH04in accordance with present invention;

Fig 15 illustrate annotated result of top 15 organisms found in BLASTX for the fungal sample AGPH04 in accordance with present invention;

Fig 16 illustrate top 13 terms in biological function category identified using Gene ontology annotation for sample AGPH04 in accordance with present invention;

Fig 17 illustrate top 13 terms in molecular function category identified using Gene ontology annotation for sample AGPH04in accordance with present invention;

Fig 18 illustrate top 13 terms in cellular component category identified using Gene ontology annotation for sample AGPH04in accordance with present invention;

Table 1: BLAST results of ITS-1, 5.8 S, and ITS-2 rDNA sequence data of AGPH#04

Table 2: Thermal Stability of Phytotoxin

Table 3: Bioassay of solvent extracted compound from AGPH04

Table 4: Effect of AGPH&04 on the growth of Parthenium

Table 5: Effects of metabolites produced by Altemaria on excised leaves of Parthenium

Table 6: Effects of Alternaria AGPH&04 on Shoot cut of Parthenium

Table 7: Host specificity testing of AGPH 04

Table 8: Testing of various formulations of AGPH 04 by Seedling bioassay

Table 9: Raw read summary

Table 10: Trimmed read summary for samples AGPH04

Table 11: KmerGenie Results

Table 12: Assembly Statistics for contigs

Table 13: BLASTX and UniProt Summary

Table 14: Annotation status

Table 15: Gene ontology terms identified in each category

DETAILED DESCRIPTION OF THE INVENTION

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The present invention relates to Parthenium control compositions comprising living fungal cells, Cell free broth, crude metabolites or purified compound of plant pathogens effective for the control of Parthenium weeds. Regarding control methods of weeds, plant diseases and insects, substantial developments have taken place in approximately the last 100 years owing to developments in chemistry. As a result, chemical control methods have replaced traditional cultivation control methods. In particular, the rapid progress of synthesized organic pesticides in the middle of the 19th century, led to increased field crop yields, improved quality, farming labor saving, etc., so that the world food production was improved rapidly. Serious problems have, however, arisen in the last several years from the use of such synthesized organic pesticides, including environmental pollution and reduced control effects, resulting from the occurrence of pathogens and insect pests with acquired pesticide resistance. In recent years, the development of synthetic pesticides has therefore apparently been oriented towards a higher activity of the pesticides. Namely, pesticides are developed these days, which are harmless to mammals and display good effects at low application rates while paying sufficient attention to residuality, environmental pollution and the like. On the other hand, there has recently been a growing interest in so-called organic agriculture systems in which crops are cultivated without pesticides. People are very concerned with the influence of synthetic organic pesticides to the human body and their influence to the natural environment as mentioned above and in recent years, the interests in biopesticides has increased enormously, so that a great deal of research and development work is under way with respect to biopesticides. The establishment of a total control system relying upon the combination of a biological control method, which makes use of a natural enemy or the like, and a cultivation or cultural control method involving the use of a cultivation system, such as crop rotation, may be mentioned by way of example. From the foregoing background, there is a keen demand for the establishment of biological control methods. Great hope is placed especially on the development of biopesticides directly using the living cells of a microorganism and microbial origin pesticides using a physiologically active substance produced by a microorganism. In the field of herbicides, extensive investigations have been carried out in recent years on mycoherbicides and microbial metabolites against weeds. Neither mycoherbicide nor microbial origin

2018267591 20 Nov 2018 herbicides are yet available against Parthenium. An object of this invention is to provide novel weed control means capable of taking over the position of synthesized herbicides. The present inventors have found certain strains of the fungus Alternaria sp. as pathogens effective for the control of Parthenium. The present invention therefore provides a weed control composition comprising living Alternaria sp. fungal cells or cell free broth, crude metabolites or purified metabolites thereof as well as a weed control method using formulated cell free culture filtrate οιAlternaria sp. The pathogens, Alternaria sp., useful in the practice of this invention exhibit a specific pathogenicity to Parthenium. The use of these microorganisms as mycoherbicide makes it possible to control Parthenium, a troublesome weed, without adversely affecting adjacent some economic crops. Further, the pathogens which are useful in the practice of this invention have been chosen from a variety of naturally occurring microorganisms and therefore are free of the potential problem of environmental pollution by synthesized organic herbicides and can be used safely. Namely, Alternaria sp. useful in the practice of this invention were found by subjecting to pure isolation pathogens, which had been collected from lesions of naturally-infected Parthenium, and then selecting, from the thus-isolated pathogens, those being pathogenic to Parthenium but non-pathogenic to economic crops like chilies, tomato and brinjal. Pathogens useful in the practice of this invention were selected by conducting both herbicidal activity and pathogenicity tests on Parthenium and chilies, tomato and brinjal plants with respect to strains isolated from naturally-infected Parthenium. As a result of a morphological and 18s rRNA identification of strains, it was found to be classified as Alternaria sp named as AGPH&04. It is deposited to NCIM with accession number NCIM 1371. The deposition were converted to depositions under the requirements of the Budapest Treaty, IMTECH to get accession number MTCC 5973.The weed control compositions of this invention, which possess the specific pathogenicity against Parthenium only, can use selected strains of Alternaria sp. As a method for using Alternaria sp. in such weed control compositions, cultured living fungal cells can be used directly as they are, or after culturing the cells, the culture resulting from germ-free filtration to use a metabolite thereof. The mycoherbicidal compositions of the invention are prepared by dispersing the cultures in suitable medium at an application rate of active agent, preferably ranging from aboutlOml of formulated culture broth in one litre of

2018267591 20 Nov 2018 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. 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.

Effective microbial weed control compositions and microbial origin weed control compositions can be produced by mass-culturing the Alternaria sp. and efficiently obtaining spores, both under asceptic conditions. When applied to the weed, these weed control compositions possess selective herbicidal activities against Parthenium 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. Pathogens Alternaria sp. useful in the practice of this invention will hereinafter be specifically described by the following examples.

Therefore, the present invention provides a herbicidal composition for controlling Parthenium sp., the composition comprising a herbicidal agent or phytotoxin from an Alternaria strain. The Alternaria strain preferably is a strain having the characterizing features of Alternaria strain AGPH#04 as deposited at IMTECH under accession number. MTCC 5973. A characterizing feature of this strain, is its 18s rRNA sequence. Sequence analysis showed 92% nucleotide similarity of the Fungi to Alternaria species but also indicated nucleotide variation of this fungus from other known Genus (Figi). In addition AGPH#04 has surprising features in respect of herbicidal activity, in particular against Parthenium sp. The Alternaria strain having the characterizing features of Alternaria

2018267591 20 Nov 2018 strain AGPH&04 most preferably is Altemaria sp. AGPH&04 deposited as MTCC 5973 or NCIM1373 . = > .

In an embodiment of the present invention, the herbicidal agent may be a hyphal or a spore inoculum, such as a conidial inoculum. The term inoculum referring to an agent comprising fungal biomass. The inoculum may be viable or non-viable. A non-viable inoculum may be obtained by subjecting a viable inoculum to an inactivating treatment as is known in the art. Heat treatment may be suitably used for inactivating an inoculum. Hyphal and/or (conidia) spore inocula may be obtained from solid or liquid fermentations. A hyphal inoculum may be selected as a hyphal suspension. Similarly a (conidia)spore inoculum may be selected from a spore suspension.

In an embodiment of the present invention, the herbicidal agent may be culture broth (fermentation broth). Within the present invention the term culture broth refers to the liquid in which the fungus was grown. For fungal growth for example PDA medium may be used. Fungal growth may commence between 4 and 9 days, such as between 5 and 8 days, preferably between 6 and 7 days, more preferably about 7 days. Culture broth may be used as it results from a liquid fermentation and may contain fungal biomass such as hyphal and/or (conidio) spore biomass. According to certain preferred embodiments the culture broth is at least partially purified. The term “at least partially purified” comprises partially purified and may be substituted for this term. A partially purified culture broth results from performing a number of purification steps that increase the contents of certain compounds of the culture broth (while removing others). A partially purified culture broth remains a mixture of compounds. For example a partially purified culture broth may be cell free culture filtrate (CFCF) for example obtained by filtering-off the fungal biomass with any known means such as microfiltratibn. A partially purified culture broth may further be obtained by (organic) solvent extraction of certain compounds from the culture broth or from a CFCF obtained therefrom. According to certain embodiments the solvent is selected from hexane, Ethyl acetate, chloroform, methanol or mixtures therefrom. The selected solvent preferably comprises hexane or Ethyl acetate and preferably is hexane or ethyl acetate. Most preferably hexane is used. When using hexane or ethyl acetate as a solvent a potent herbicidal agent is obtained as is further detailed in the examples. The invention therefore further relates to a herbicidal

2018267591 20 Nov 2018 agent obtainable by organic solvent extraction, in particular a hexane or ethyl acetate extraction, of CFCF of the Alternaria strain used. The solvent extract may have the elution profile presented in figure 2, or a similar profile, on a HPLC, Cl8 Reverse phase column. The use of hexane for solvent extraction is most preferred. Preferably by using methanol: water (0-100% gradient) as solvent system. The herbicidal agent according to the invention will comprise a number of secondary metabolites. “A number of’ within the present invention should be construed as meaning one or more) such as a plurality, for example 2-10 such as, 3-9, 4-8, 5-7, or 6. Secondary metabolites in the herbicidal agent may be further purified. According to certain embodiments the secondary metabolites are purified to > 80% purity, such as >85%, >90%, >95%, >99% up to 99.9 % purity.

The compositions and methods of the invention include the provision of application of cell free culture filtrate or their metabolites to produce a novel herbicidal agent. Herbicide compositions may be prepared as a liquid formulation by suspending the broth, partially purified broth, such as 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 comprises a cell free broth.

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. '

According to preferred embodiments in the formulation a surfactant, such as a Tween, for example Tween 20, or Tween 80 is used. As is shown in the experimental section such a combination results in a surprising increase of the activity of Parthenium control.

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According to other preferred embodiments in the formulation an oil, such as a mineral oil, for example paraffin oil, or a vegetable oil, such as coconut oil is used. As is shown in the experimental section such a combination results in a surprising increase of the activity of Parthenium control. Antibiotics may also be included Antibiotics (antimicrobials) may be selected from Streptomycin, Ampilox, Azithromycin may be selected from these antibiotics.. In general formulating agents can be added in an amount of 0.2-0.9 % w/w, such as 0.3-0.8%, 0.4-0.7%, 0.4-0.6%, such as about 0.5% w/w to the herbicidal agent. Antimicrobials may be added in an amount of 0.05-0.5% w/w such as 0.06-0.4 %, 0.07-0.3%, 0.08-0.3%, 0.09-0.3 %, 0.1-0.3 % such as about 0.2% w/w to the herbicidal agent.

A further aspect of the invention relates to a method of controlling weeds, in particular Parthenium sp. The method includes applying the herbicide composition to the Parthenium plants. The herbicide composition can be applied by spraying a solution of fungal herbicidal agents, such as 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. Also the method of the present invention may be used in addition to or in conjunction with other control measures.

As such the method is for suppressing or preventing (controlling) growth of Parthenium sp. and the method comprises contacting the Parthenium plants, preferably Parthenium hysterophorus plants, with the a composition of the invention. Details of the technical features of the composition of the invention have been discussed above. As the skilled person will understand the composition of the invention will be used in an effective amount sufficient to bring an effect, in particular control (reduction or inhibition) of growth of Parthenium plants. It is within the ambit of the knowledge and skill of the skilled person to determine effective amounts. According to certain embodiments effective amounts may be in the range of 10ml of formulated cell free culture broth to 1 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. In certain embodiments of the present invention, the contacting of the Parthenium plants with the composition is achieved by applying the composition on the plants for

2018267591 20 Nov 2018 example by spraying. In other embodiments of the present invention, the contacting of the Parthenium plants with the composition is achieved by mixing the composition in soil where Parthenium is or may be present as seeds and/or roots.

A further aspect of the invention relates to the use of an Alternaria strain, preferably a strain having the characterizing features of Alternaria strain AGPH#04 as deposited at IMTECH under accession number MTCC 5973 for producing a herbicidal agent effective for controlling growth of Parthenium sp, in particular Parthenium hysterophorus. The technical features of the herbicidal agent have been discussed above in connection to the discussion of the composition of the invention. Similar to what is discussed for the composition of the invention, the herbicidal agent may be selected from a hyphal inoculum, a spore inoculum, preferably a conidial inoculum, culture broth, preferably at least partially purified culture broth metabolites. Yet a further aspect of the invention relates to a herbicidal agent obtainable by a method comprising solvent extraction, preferably hexane or ethyl acetate extraction, of cell free culture filtrate (CFCF) of a Alternaria strain, preferably a strain having the characterizing features of Alternaria strain AGPH#04 as deposited at IMTECH under accession number MTCC 5973. Most preferably the Alternaria strain is strain AGPH#04 as deposited at IMTECH under accession number MTCC 5973 or a strain derived therefrom. In the method the CFCF is provided, preferably by culturing the selected strain in a liquid culture and producing a CFCF from the culture broth. Next a (organic) solvent extraction, preferably hexane or ethyl acetate extraction is performed on said CFCF. The solvent extraction may be executed according to any known methods. The strain was grown at 28°C±1°C under in one litre flasks containing suitable medium. The sterile media was inoculated with 1 ml of a spore suspension of fungus, shaken gently, and incubated as standing cultures for 14 days. The culture filtrates were collected by successive passage through Whatman No.l filter paper, and a sartorius membrane filter (0.20 pm). The culture filtrate which has been passed through the sartorius membrane filter. Cell free culture filtrate extracted with chloroform to get to solvent extract. The chloroform extract sterile filtrates were stored at 4°C depending on assay or fractionation. High Performance Liquid Chromatography (HPLC) was performed using methanol: water (0-100% gradient for 30 minutes) as solvent system in C18 reverse phase column.

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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

Isolation and identification of host specific effective mycoherbicidal strain:

A th=mgh systematic and periodical survey of various regions of Andhra Pradesh was made to collect diseased specimens. They were dried by pressing method and deposited in the Laboratory for recovery and isolation of strains. Tissues from diseased portion of the weed cut down into about 1 mm pieces sterilized and transferred to PDA Petri plates. Plates were incubated at 28°C in BOD incubator and examined regularly. As soon growth appeared transfer to slant. Total 442 number of strain were collected. Only 76 were selected for trails. Detailed taxonomic descriptions were prepared. The fungal pathogens were identified with the help of available literature. For preliminary pathogenicity test conidia of fungi were harvested by flooding Petri-dishes of seven days old cultures with sterile distilled water. The spore’s concentration was adjusted with haemocytometer to 2.5xl0⁶ spores/ml. The potential of isolates were assessed using detached leaf bioassay or shoot cut bioassay method. The strains showing LAD (Leaf area damage between 80 to 100%, has taken for second trails. Pure cultures of the recovered fungi were prepared from single conidium and maintained on half strength PDA slants as stock cultures. The isolated fungi were identified to the genus based on their conidial morphology and growth characteristics on various growing media. Further identification and characterization were done on isolate that was confirmed to be highly pathogenic to P. hysterophorus by applying Koch’s postulates. Cultures grown on PDA as well as sporulating on infected plants in the greenhouse were used for taxonomic characterization of the conidia.

Few genera of fungi were consistently isolated from the diseased P. hysterophorus, however, only one fungus fulfilled the Koch’s postulate. The fungus was highly pathogenic on diseased P. hysterophorus. The symptoms produced on P. hysterophorus when inoculated with this fungus under green house conditions were similar to those seen in the field. In PDA plates after 7 days of incubation colony growth was uniform and faster, with average diameter of 66.8 mm and 66.1 mm, respectively. Isolates of fungi

2018267591 20 Nov 2018 form unbranched or poorly branched conidial chains on short unbranched conidiophores. Conidia are dark in colour, multicellular with transverse and longitudinal septae. They are of different size regarding the place of formation in the chain. Based on these characteristics, the tested isolates were determined as Alternaria sp. as per Singh, 2007 and designated with lab code AGPH04.

Similar observations was observed in second trails with Parthenium shoot cut bioassay and on the basis of these observations, it can be concluded; that Alternaria sp. has enormous potential as herbicide against Parthenium weed.

Alternaria isolate was isolated from infected Parthenium leaves plants exhibiting symptoms of leaf disease. During survey to Village Adavi Ramanpalam, PenuballiMandal, Khammam District, Andhra Pradesh, Parthenium leaves infected with pathogenic fungus Alternaria sp AGPH#04 is a case in point to develop as herbicide. The isolates were grown on the PDA medium and stock cultures of these were maintained at AGBIO Systems Laboratory and also deposited in the NCIM, Pune, India and given an accession number NCIM 1371.This strain has also been deposited at IMTECH, Chandigarh, India tinder the Budapest treaty under accession number. MTCC 5973. The strain has identified as Alternaria alternata.

Alternaria sp. grow rapidly and the colony size reaches a diameter of 3 to 9 cm following incubation at 25°C for 7 days on potato glucose agar. The colony is flat, downy to woolly and is covered by grayish, short, aerial hyphae in time. The surface is greyish white at the beginning which later darkens and becomes greenish black or olive brown with a light border. The reverse side is typically brown to black due to pigment production. The macroscopic figures of Alternaria sp AGPH04 is mentioned in Fig 1.

Microscopic morphology Alternaria sp. has septate, dark hyphae. Conidiophores are also septate and sometimes have a zigzag appearance. They bear simple or branched large conidia (8-16 x 23-50 pm) which have both transverse and longitudinal septations. These conidia may be observed singly or in acropetal chains and may produce germ tubes. They are ovoid to obclavate, darkly pigmented, muriform, smooth or roughened. The end of the conidium nearest the conidiophore is round while it tapers towards the apex. This gives the typical beak or club-like appearance of the conidia. The microscopic figures of Alternaria sp. is mentioned in Fig 2.

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EXAMPLE 2:

Sequence homology and phylogenic analysis

Alternaria sp AGPH#04 are potential, highly hosts specific mycoherbicide for control of the Parthenium weed (Table 1 & Fig 3). The fungal isolates were 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. The 95% similarity has been shown by Alternaria alternata AF21879. This unique ITS sequence of the two isolates provides strong evidence to consider these pathogens as new from speicalis that will facilitate and encourage the introduction and acceptance by regulatory authorities for practical field application.

Table 1: BLAST results of ITS-1, 5.8 S, and ITS-2 rDNA sequence data of AGPH#04.

Organism	GenBank Accession(s)	% identity (bp)
Alternaria alternata AF21879.1	AF21879	95
Alternaria cichorii	AM237286.1	95
Alternaria jesenskae	AM237084	95
Alternaria multirostrata	AM237487 '	95
Alternaria passiflorae	AM237288	94
Alternaria sp	AY 154699	98
Alternaria sp	AY 154698 /	98
Alternaria tenuissima	AY154712 :	98
Alternaria tenuissima	AY154711	98

EXAMPLE 3:

Production, extraction and characterization of phytotoxic coihpound.

Alternaria sp was grown at 28° C±1°C under in one litre flasks containing 250 ml modified Richard medium with 1% host leaves extract. The sterile media was inoculated with 1 ml of a spore suspension of Alternaria sp, shaken gently, and incubated as standing cultures for 14 days. The culture filtrates were collected by successive passage through Whatman No.l filter paper, and a Sartorius membrane filter (0.20 μηι). The culture filtrate which has been passed through the Sartorius membrane filter. The sterile filtrates were stored at 4°C depending on assay or fractionation. For the phytotoxin

2018267591 20 Nov 2018 extraction, cell free filtrate was extracted successively with hexane, Ethyl acetate, Chloroform and methanol. The scheme for organic solvent extraction has presented in fig 4. Phytotoxic compounds were detected both in the hexane fraction. Hexane fraction were collected and evaporated. This fraction named as crude filtrate.

HPLC characterization of Phytotoxin:

The extraction procedure for toxin was followed as given above. High Performance Liquid Chromatography (HPLC) was performed using methanol: water (0-100% gradient for 30 minutes) as solvent system in C18 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 . 5).

The 9.5 gm crude brown oily residue has extracted with Hexane solvent from 1 litre of AGPH04 Culture broth (Fig. 6)

Thermal Stability of Phytotoxin

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 ofAGPH#04, was subjected to different temperature treatment viz. 40, 50, 60, 100 and 120°C. 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.

Data presented in table 2 showed that the phytotoxicity of fermented broth of AGPH 04 did not change and was stable at 40, 50, 60 and 100°C.

Table 2 Data presented in table showed that the phytotoxicity of fermented broth of AGPH 04

AGPH#0 -	'2 7	t<3 <	. >	;2 i	'3 ’	f5 d	r2 ]	p -	i3	P 7	t3 )(	;.5 r	;o -	-0 ’	-0 -
4	400	2		500(			600(	-t		100C	IC		120(	IC
Control a	'2 -	‘3 -	-5 -	2 -	3 -	'5 -	-2 -	-3 -	'5 -	-2	—1 J	-5 -	‘2 -	-3 -	‘5 ’
Control b	0	0	0	0	0	0	0	0	θ	0	0	0	0	0	0
	t	t	t

Phytotoxicity scale of 0 to 5 with 0 = no control, 5 = complete leaf necrosis

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HPT = hour post treatment

Ctrl A: Room temperature

Ctrl B: Broth medium.

EXAMPLE 4:

Phytotoxicity test of various organic solvent extracts:

Detached leaf of Parthenium with solvent extracted metabolites has tested for phytotoxicity. More phytotoxicity was detected in the hexane and ethyl acetate fraction (Table 3). Hexane has showing 100% toxicity so hexane has selected as best solvent for metabolites extraction.

Table 3: Bioassay of solvent extracted compound from AGPH04

Items	Phytotoxicity
Control	Methanol extract	Hexane extract	Chloroform extract	Ethyl acetate Extract
Detached leaf of Parthenium	0	+ .	++++	+	+++

Phytotoxic activity indexing was on a scale

0= 0 treated with water, no lesion; + = 5% diam; ++= 25%; +++= 75% ++++= 100%,

Phytotoxicity of CFCF, Crude and HPLC purified phytotoxic compound on Parthenium Seedling

Parthenium seeds were 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. Parthenium plants were used in these experiments. Following inoculation, plants were incubated on greenhouse benches under conditions as described above. Three replicates of weed were used for each treatment. The experiment was repeated four 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 4.

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In Parthenium 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 4: Effect of AGPH#04 on the growth of Parthenium seedling

Source	Toxin used (μΐ)	Phytotoxicity
Cell free culture filtrate	10.0	+
Crude filtrate	5.0	+
Partially purified	1.0	+
Control	Distilled water	- ' 1

- = no phytotoxic effects, += phytotoxic effects.

EXAMPLE 5:

Phytotoxicity of CFCF, Crude and HPLC purified phytotoxic compound on Parthenium detached leaves.

Detached Parthenium 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 micropipettes. 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 5.

Table 5: Effects of metabolites produced on excised leaves of Parthenium

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

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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:

Phytotoxicity of CFCF, Crude and HPLC purified phytotoxic compound on Parthenium shoot cut

Shoot cut Parthenium 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 Parthenium were incubated under continuous or 12 h light. The phytotoxic effects on the treated shoot cut of Parthenium 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 6.

Table 6: Effects of Alternaria AGPH#04 metabolites on Shoot cut of Parthenium

Source	Concentration (μΐ/ml)	Phytotoxicity
Cell free filtrate	100	+ ! '
Crude filtrate	10	+ ’ '
Partially purified	1	+
Control	NIL	-

The phytotoxic damage was evaluated visually for 10 days. -= no phytotoxic effects, += phytotoxic effects. ’

EXAMPLE 7:

Host specificity of strain

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Four weeds, three crops and one from similar families were used for testing host specificity of strain AGPH#04. 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. Similar family of Plant, Chrysanthemum was also treated as detached 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 7.

Table 7: Host specificity testing of AGPH 04

Plants testes	Seedlings	Detached leaves
Chilly	-	NIL
Tomato	-	NIL
Brinjal	- ' '	NIL
Parthenium	NIL	+++
Lantana	NIL	- '
Hyptis .	NIL	-
Cassia tora	NIL	-
Chrysanthemum (Asteraceae family)	NIL

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

As a result of the test, Alternaria ^.useful in the practice of this invention was found to have absolutely no pathogenicity against crops and other weeds.These microorganisms of this invention were hence recognized to be usable as mycoherbicides .

EXAMPLE 8

Formulation Study:

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Formulation refers to the process of blending of secondary metabolite or microbe with inert carriers to alter their physical characteristics to enhance its shelf life and field performance. Formulation involves the use of formulants which include: adjuvants, surfactants, wetting agents, spreaders and preservative. The use of formulative for biological control is a relatively new application of technology that is well known to the chemical herbicide industry. Adjuvant increases the efficacy of post emergence herbicides by increasing the wettability of the target surface as they reduce surface tension. Adjuvants are also known to enhance penetration. In order to investigate the compatibility of the phytotoxin produced by the test fungus with various formulants were tested. To test the compatibility of the toxin synthesized by the pathogen seven formulating agents namely, Tween-20, Ek Bond, mineral oil, mustard oil, coconut oil, groundnut oil, paraffin oil, glycerol were used. Formulating agents were added @ 0.5% to the CFCF containing phytotoxin with antimicrobial 0.2% and compatibility was determined by seedling bioassay.

Data represented in Table 8, shows the compatibility testing of various formulants @ 0.5% on Parthenium seedling bioassay. The phytotoxic damage studies clearly show that the most compatible formulant was Paraffin oil with preservative followed by coconut oil, Ek bond, Tween-80, Tween-20, Lipase and glycerol. Average effect was shown by mineral oil. Oils are used as additives for a variety of reasons such as reducing vapour loss of herbicide, enhancing the performance of herbicides. Traditionally, spray formulations have incorporated petroleum-based oils. Petroleum oil based adjuvants are known to enhance the phytotoxicity of herbicides. Thus formulation with paraffin oils can enhance absorption, translocation and phytotoxicity of herbicides. In order to investigate the compatibility of the phytotoxins produced by the test fungus various formulants were tested. On the basis of results obtained above, it can be concluded that the secondary metabolites of AGPH04 possess high herbicidal potential with paraffin oil and can be developed as potential herbicides for the management of Parthenium weed.

Table 8: Testing of various formulations of AGPH#04 by Seedling bioassay.

SN	Formulants	Parthenium seedling
		Plant disease rating

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		24 hpt	48 hpt	72 hpt.
1	CFCF + antimicrobial	2	2	2
2	CFCF + Lipase	2	2 .	2
3 .	CFCF +Coconut oil	2	2 ;	2
4	CFCF + Tween 20 + antimicrobial	3	3 1	4
5	CFCF + Paraffin oil + antimicrobial	4	5	5
6	CFCF +Mineral oil + antimicrobial	2	2	2
7	CFCF +Glycerol + antimicrobial	2	2	2 .
8	CFCF +Mustard oil+ antimicrobial	2	2 .	2
9	CFCF + EK bond + antimicrobial	2	2	3
10	Control a	0	0 :	0
11	Control b	0	0 ,	0

Control a- growth medium Control b- Sterilized Distilled Water; Amount of CFCF containing phytotoxin employed= 10 ml/plant

PLANT DISEASE RATING (PDR)

0-1— slight curling & wilting; 1 -2=slight chlorosis; 2-3=marked chlorosis, slight necrosis; 3-4=high necrosis and marked chlorosis; 4-5=acute necrosis and marked chlorosis; 5=acute chlorosis and acute necrosis leading to death of seedling.

Example 9

Whole Genome Study

Plant fungal pathogen in the genus Alternaria infects a remarkable range of host plants and is major causes of agricultural yield losses. To gain a richer understanding of the molecular determinants of virulence and the evolution of pathogenicity, we have performed whole genome sequencing of Alternaria sp.

Alternaria sp DNA was extracted by Scigenom developed protocol and the sequencing library was prepared using Illumina paired end DNA sample prep kit. Sequencing was performed using Illumina Genome Analyser. Short reads were assembled de novo using velvet and assembly quality was improved by a pipeline including two alternate assemblers Edena and Minimus.

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Alternaria sp AGPH#04 (NCIM 1371) was sequenced by a whole genome shotgun approach using the Illumina Genome Analyzer. Fastq quality check, De novo assembly of genome study, Gene prediction, ORF annotation and Blastx search performed.

Fastq quality check and filtering results are shown in Table 9

Table 9: Raw read summary

Sample	AGPH04
Number of paired end reads	19,419,816
Number of bases (Gb)	3.88
GC%	52.09
Percentage of data>=Q30	80

Fastq quality check involved checking of quality parameters for the sequence obtained from sequencer. Base quality score distributions, average base Content per read and GC distribution in the reads were performed for an input fastq file. The base quality score distribution, the quality of left (Rl) and right (R2) end of the. paired end read for the sample is shown in fig 7, 8.

The x- axis represents sequencing cycle and y axis represent the phred quality score of biases. It was observed that average base quality was above Q30 (error probability >= 0.001) for more than 80% of bases. Base composition distribution of left and right end of the paired end is shown in fig 9, 10.

The x-axis represents sequencing cycle and y axis represents nucleotide percentage. We observed bias in the base composition towards the beginning of reads. Biasing in sequence composition is generally observed in NGS experiments. The GC distribution of left and right end of the paired end read sequence were performed (Fig 11, 12).

The x-axis represents average GC content in the sequence and y axis represents total percentage of reads. The average GC content of the reads in the sample followed a normal distribution. The fastq files were trimmed before performing Denovocontigs assembly. Scigenom scientists have trimmed three bases from the beginning and two bases from the end of the reads. Summary of the trimmed reads are provided in Table 10. Table 10: Trimmed read summary for samples AGPH04

Sample ^: AGPH04 ^:

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Number of paired end reads	19,419,816
Number of bases (Gb)	3.69
Read length	2x 95bp

Further, filtered out reads that were contained with illumine adapter. Kmer Genie used to predict the optimal k value and assembly size. KmerGenie results are recorded in Table

11.

Table 11: KmerGenie Results

Sample	AGPH04
Predicted best k	65
Predicted Assembly size	32,719,345

Various assembled independent contigs are used to integrate the contigs. For AGPH04 the final assembly had 273 contigs and the longest contig was 3882817bp long. The assembly statistics is provided in Table 12.

Table 12: Assembly Statistics for contigs

Assembler	Min	Max	Mean	N50	Total contigs	Sum of length of all contigs
AS	203	3882841	73243.14	1400142	449	32886169
Edena	142	788906	117378.1	237270	278	3263112
SOAPdenovo	66	416352	17506.48	159880	1875	32824645
Velvet	129	393194	24868.22	117758	1318	32776315
MaSuRCa	300	658306	69148.87	127630	475	32845711
C1SA	85	3882817	120775.64	1632136	273	32971749

For gene prediction and Annotation, the predicted ORFs from the contigs for the fungal sample AGPH04 using Augustus method. Compared to gene prediction for AGPH04, we have used trained data from Aspergillusnidulans and Neurosporacrassa to predict ORFs independently. We found 14, 915 unique ORFs in sample AGPH04 from these predictions. The predicted ORFs were annotated using Scigenom in house pipeline (CANoPIContig Annotator) for de novo assembly. The steps for annotation of ORFs are

2018267591 20 Nov 2018 comparison with NCBI database using BLASTX program, Organism annotation, gene and protein annotation to the matched ORFs, Gene ontology annotation and pathway annotation.

The predicted ORFs were compared with NCBI non redundant protein database using BLASTX program. Matches with E value <=le‘⁵ and similarity score>=40 were retained for further annotation. BLASTX summary is provided in Table 13. For Sample AGPH04 overall we found 14, 382(96.43%) of predicted ORFs have at least one hit in NCBI database.

Table 13: BLASTX and UniProt Summary

Categories	AGPH04
Number of ORFs	14,915
Number of ORFs with significant BLASTX match	13,828
Number of ORFs with UniProt annotation	13,759

The BLASTX search E value distribution is provided in Fig 13. Around 89% of the ORFs found using BLASTX for the sample had confidence level of at least le-50, which indicated high protein conservation. The BLASTX similarity score distribution for sample is shown in Fig 14. 83% of the predicated ORFs found using BLASTX had similarity of more than 60% at protein level with the existing protein at NCBI database in Sample AGPH04.

For organism annotation the top BLASTX hit of each ORF was studied and the organism name was extracted. The top 15 organism found in the annotation for the sample shown in Fig 15.

Majority of the top BLASTX hit belong to Pyrenophorateres f teres.

For gene and protein annotation, the predicted protein from BLASTX were annotated against NCBI, UniProt pathway and other databases. Among the total significant BLASTX hit ORFs 13, 759 were annotated using UniProt database. For remaining BLASTX hit NCBI predicted protein annotation were provided. The complete annotation were divided into following three categories (Table 14).

Claims (15)

1. A herbicide composition comprising a herbicidal agent from strain of an Alteinaria_spQc\es having the characterizing features of strain Alternaria species AGPH&04 as deposited at NCIM and IMTECH under accession number NCIM 1371, MTCC5973respectively

2. A herbicide composition as claimed in claim 1, wherein the herbicidal agent is selected from a hyphal inoculum, a spore inoculum, preferably a conidial inoculum, culture broth, preferably an at least partially purified culture broth, a secondary metabolite.

3. A herbicide composition as claimed in any of the claims 1-2, further comprising a number of additives, such as conventional formulation additives for examples surfactants, solvents, carriers and diluents.

4. A method for controlling Parthenium weeds, said method comprising contacting the Parthenium weeds with a_herbicide composition according to any of the claims 1-3, wherein said contacting preferably is achieved by applying the composition on the weed such as by spraying.

5. A method as claimed in claim 5, wherein the Parthenium weeds are from the species Parthenium hysterophorus.

6. A method as claimed in claim 5, wherein the herbicidal agent is selected from a hyphal inoculum, a spore inoculum, preferably a conidial inoculum, culture broth, preferably an at least partially purified culture broth, a secondary metabolite.

7. A method as claimed in any of the claims6, wherein the herbicidal agent further comprises of a number of additives, such as conventional formulation additives, for example surfactants, solvents, carriers, diluents.

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8. A method of identification of microbial culture using 18S -ITS sequence of rRNA based molecular technique.

9. A method for first study to perform whole-genome sequencing, and comparative genomics of weed pathogenic fungi.

10. A method for identifying important core genes and attempts to examine expression differences for organisms with very different host ranges.

11. A strain of Alternaria sp having the characterizing features of strain Alternaria sp. AGPH#04 as deposited at NCIM and IMTECH under accession number NCIM 1371, MTCC5973 respectively.

12. Herbicidal agent obtainable by a method comprising hexane or ethyl acetate extraction of Cell Free Culture Filtrate of Alternaria sp. preferably a strain having the characterizing features of Alternaria sp. AGPH&04 as deposited at NCIM and IMTECH under accession number NCIM 1371, MTCC 5973 respectively.

13. Use of an Alternaria strain, preferably a strain having the characterizing features of Alternaria strain AGPH04 as deposited at NCIM and IMTECH under accession number NCIM 1371, MTCC 5973, respectively for producing a herbicidal agent effective for controlling growth of Parthenium weed.

14. Use according to claim 12, wherein the herbicidal agent is selected from a hyphal inoculum, a spore inoculum, preferably a conidial inoculum, culture broth, preferably an at least partially purified culture broth, secondary metabolites.

15. Use according to any of the claims 12-13, wherein the herbicidal agent is obtained from a hexane or ethyl acetate extraction of Cell Free Culture Filtrate of the selected Alternaria sp.