A SCRIPT OF P.G.

SEMINAR
ON
ROLE OF BIOHERBICIDE: A NEW ERA FOR WEED CONTROL

Submitted by:

Mr. Pravin Shamsing Deore

Reg. No- J4 – 00075-2005
M. Sc. (Agri.) Student
J.A.U., Junagadh.

Major advisor: Minor advisor:

Dr. V. D. KHANPARA Dr. B. A. GOLAKIYA
Professor and Head, Professor and Head,
Dept. of Agronomy, Dept. of Biochemistry,
College of agriculture, College of agriculture,
JAU., Junagadh. JAU., Junagadh.

Submitted to:

Dr. R. L. KALAWADIA
Convener of P. G. seminar
Professor (P) and Head
Department of Agril. Statistics,
College of Agriculture,
J.A.U., Junagadh.

DEPARTMENT OF AGRONOMY,
COLLEGE OF AGRICULTURE,
JAU, JUNAGADH.
Introduction:
Biological control of weeds by using plant pathogens accepted as a practical, safe,
environmentally beneficial, weed management method applicable to agroecosystems
(Charudattan, 2001). Bioherbicides are pathogens cultured artificially and made available
in sprayable formulations; just like a chemical [Link] pathogen selected for the
purpose is usually from the native place of the weed, but it could also be from other
places. The bioherbicides are also some times called mycoherbicides.
The Active ingredient in a bioherbcides is a living microorganism and it is applied
in inundative doses of propagules. Microorganisms such as fungi, bacteria, viruses are
used but most commonly use microorganism is a fungus.

Bioherbicide philosphy of weed control:

1)Bioherbicide remains active only on the current weed populations, without any chance
of cyclic perpetuation of the weed (or of the bioagent); each new flush of the weed thus
requiring retreatment with it.
2) Bioherbicide can be developed for selective control of weeds in a crop just like any
other selective herbicide, which is not the case with classical philosophy bioagents.
3) The development of herbicides is of great interest to industrialists since it involves
every season requirement of the product for field use. In variancce with it, the classical
biological control approach has no incentive to the private, profit-oriented organizations;
it must depend solely upon public sector support.

Why Using Bioherbicides:

1) High yield losses still occur despite availability of synthetic pesticides.
2) Herbicide resistant weed populations have developed.
3) Synthetic pesticides may have detrimental effects on non target organisms.
4) Large areas where herbicide application not possible or not effective.
5) Potential environmental impact or harm.
6) Ground water contamination.
7) Damaging effects of herbicides.

The Bioherbicide Advantages:

 Bioherbicide can be readily integrated into weed management systems.
 Bioherbicides are often compatible with synthetic herbicides.
 Organically acceptable.
 Low maintenance and management.
 Can be very specific.
 Effective area not limited.

Ideal Characteristics of a Bioherbicide:

 Produce abundant and durable inoculum in culture.
 Target specific.
 Genetically stable.
 Capable of killing a significant portion of the weed population
under a variety of environmental conditions (weed densities).
Approaches for Microbial Weed Control:
 Classical
 Inundative
 Augmentation

Classical Approach:
1. The classical or inoculative approach involves the importation and release of
one or more natural enemies that attack the target weed in its native range, in to
areas where its natural enemies are absent.
2. The classical approach has been used almost exclusively for the control for the
extensive infestation of perennial weeds in relatively undisturbed environments
such as aquatic and range land.
3. The objective of classical approach is generally not eradication of the weed
species, but self-perpetuating regulation of the weed population at
acceptable low levels.

Inundative or Bioherbicidal Approach:

1. The bioherbicide approach in which a natural enemy is used within its native
range to control a native or naturalized weed.
2. Bioherbicide have been targeted towards a wide range of weed problems,
including weeds of cropping systems.
3. Consequently, bioherbicides often must be effective in disturbed environments,
delivering economically valuable weed control within specific, short time
frame.

Augumentative Approach:
1. This approach falls into the category of periodic release and can be regarded as
midway between the classical and the inundative approach.
2. The pathogen may be native or naturalized in their respective region causing
endemic disease.
3. They can be self-disseminate causing certain level of self-sustaining epidemics
after inoculation.
4. The amount of inoculum is required larger than for inoculative approach and
less than the inundative strategy.
5. The area that needs to be inoculated is also larger.
Bioherbicides that have been registered and their current status:

Where and when Product and pathogen Target weed Status

USA:1981 DeVine®: Strangler vine Commercially used
Phytophthora (Morrenia
palmivora odorata) in citrus
orchards
USA:1982 Collego™: Northern joint Commercially used
Colletotrichum vetch
gloeosporioides f. sp. (Aeschynamene
aeschynomene virginica) in rice
and soybean
USA:1983 CASST™: Alternaria Sickle pod and No longer available
cassiae coffee senna due to lack of
(cassia spp.) in commercial backing
soybean and
peanut
USA:1987 Dr BioSedge: Puccinia Yellow nutsedge Registered, but
canaliculata (cyperus product failed due to
esculentus) in
uneconomic
soybean, s. cane, production system &
maize, potato & resistance in some
cotton weed biotype
Canada:1992 BioMal®: Round leaved
Not commercially
Colletotrichum mallow (Malvae available at present,
gloeosporioides f. sp. pusilla) in wheat,but recently taken on
malvae lentils & flax by a new finacial
backers who is
exploring market
apportunities. Can be
made on request
South Stumpout™: Acasia species in Still available for sale.
Africa:1997 Cylindrobasidium native vegetation
leave & water supplies

Netherland:1997 Biochon™: Woody weeds,

Available until end of
Chondrostereum e.g. black cherry
2000.
purpureum (prunus serotina)
marketing/production
in plantation
stopped due to low
forests. sales & regulatory
concerns
Japan:1997 Camperico™: Turf grass (poa Probably
Xanthomonas annua) in golf commercially
campestris pv. poae courses available
USA:2002 Woad Warrior: Dyers woad Registered, but never
Puccinia (Isastis tinctoria) commercially
thlaspeos in farms, available due to lack
rangeland, waste of commercial
areas and backers.
roadsides
Canada:2004 Chontrol™ = Alders, aspen & Commercially
Ecoclear™: other hard woods. available
Chondrostereum In rice of way and
purpureum forest
Canada:2004 Myco-Tech™ paste: Decidious tree Commercially
Chondrostereum species in rights available
purpureum of way and forest

USA:2005 Smolder: Alternaria Dodder species in Only just registered.

destruens agriculture, dry Company planning to
bags & do more field trials
ornamental and then market it in
nurseries 2007
Website: [Link]

Steps in Bioherbicide development:

The development of biological herbicide involves three major phases or stages:
1) Discovery
2) Development
3) Deployment

1) Discovery:
The discovery phase involves Collection of diseased plant material:-
 Isolation of the causal organism.
 Demonstration of Koch's postulates.
 Identification of the pathogen, culture of the pathogen on artificial media.
 Maintenance of the pathogen cultures in short-term and long-term storage.

2) Development:
The development phase involves:-
 Determination of optimum conditions for spore production.
 Determination of optimum conditions for infection and disease development.
 Determination of host range and elucidation of mechanism of action of the
pathogen.

3) Deployment:
Involves close collaboration between non industrial and industrial sectors through
 Formulation
 Scale-up
 Field evaluation
  Marketing stages of commercialization of new bioherbicide product.

Bioherbicide Production Methods:

In general, three methods have been evaluated for the production of bioherbicides
1) The use of living host plants.
2) Solid substrate fermentation.
3) Liquid culture fermentation.
Adequate production of many potential bioherbicides can be achieved only by
using living plants (rust fungi) or solid substrate fermentations (Alternaria spp.) these
methods are to costly for commercial use. At present, liquid culture fermentation is
the most economical method for producing microbial biocontrol agents.

Formulations for improvement of Mycoherbicides:

A. Liquid based formulations
B. Solid based formulation
A. Liquid based formulations:

Weed host Pathogen Formulation

Velvetleaf Fusarium lateritium Water+Tween-20 surfectant

Velvetleaf Colletotricum coccodes Water+sorbitol (0.75%)

Nothern jointvetch Colletotricum Component A: dried pores

gloeosporioides Component B:rehydrating agent+
surfectant
Field bind weed Phomopsis convovulus Water+gelatin (0.1%)

Sicklepod Alternaria cassaie Water+nonxynol surfectant (0.04%)

Hemp sesbania Colletotricum truncatum Water+nonxynol surfectant (0.02%)

(Schw.)
Stranglervine Phytophora Commercial
palmivora(Bult.) formulation;chlamydospores in
water
B. Solid based formulations:

Weed host Pathogen Formulation

Velvetleaf Fusarium laterium Sodium alginate-kaolin

granules

Spurred anoda Alternaria macrospora Vermiculite

Texas gourd [Cucurbita Fusarium solani Fungus-infested oats,

texana (Scheele) Gray] cornmeal/sand;sodium
alginate-kaolin granules

Marijuana (Cannabis Fusarium oxysporium Fungus infested wheat straw

sativa L.) var canabis

Hemp sesbania Colletotricum Fungus-infested wheat gluten

truncantum (PESTA)

Sicklepod Fusarium oxysporum

Schlete

Fungi:
Most fungi produce spores and thus a high inoculum level can be readily
produced on infected grain or by mixing fungal propagules in inert carriers.
Bacteria:
Very few bacteria produce endospores and thus, bacterial formulations are
problematic.
Viruses:
Some are quite amenable to drying and hence, would be easy to formulate.

Microbial Agents for Weed Control:

 Fungi

 Bacteria

 Viruses

A) Fungi:
Some Commercial Mycoherbicides in Use Abroad

Product Content Weed controlled

De-vine A liquid suspension of fungal spores of Straglevine (Morrenia
Phytophthora palmivora Butler. It odorata) in citrus
causes root rot in the weed orchards

Collego Wettable powder containing fungal Jointvetch (Aeschynomone

spores of Colletotrichum glosporiodes sp) in rice fields. The
Penzig & Sacc. Sp. aeschynomene bioherbicide causes stem
and leaf blight in the weed
Bipolaris A suspension of fungal spores of Johnson grass (Sorghum
Bipolaris sorghicola halepense)
Biolophos A microbial toxin produced as Non-specific, general
fermentation product of Streptomyces vegetation
hygroscopicus
Gupta, 2001
1) Phytophthora Palmivora:-
 A pathotype of Phytophthora palmivora has been formulated as a liquid
suspension of clamydospores for control of stranglervine in Florida citrus under
the trade name Devine*.
 This organism was developed by Ridings (1986) and was the first mycoherbicide
to be registered.
 The fungus, a native of Florida, kills seedling and adult Stranglervine, a native of
South America.

Basic Information:
 The application rates of DeVine is 1.2 L/ha. The Fungus Can be applied from
May to September after the weed has germinated or is actively growing.
 DeVine is sprayed on the soil surface at the base of the vine using a boom
sprayer.
 With sufficient moisture, the spores of DeVine infect the top 1/3-inch of the soil
and main stem root.
 Within 6-10 weeks the vine begins to die depending on the size and maturity of
the vine.

Effects and Results of DeVine Myco-Herbicide on Florida Citrus Groves:

 Properly applied DeVine kills established and germinating vines for one year or
more.
 DeVine can be control up to 90% or greater.
 The fungus continues to grow down in to the root tissue and eventually girdles the
stem. The disease process is very dependent on high soil moisture.
 About two weeks after infection, wilting leaves will appear.
  After the leaves wilt and drop, sloughing of the tissue from the main root leader
and general root rot will be visible. ([Link]).

2) Colletotricum gloeosporoides f. sp. aeschynomene:-

 Spore of this anthracnose fungus have been sold under the trade name Collego*
for control of nothern jointvetch in rice and soybeans.
 This product is wettable powder containing dried spores produced by liquid
fermentation and applied at rate of 3.1 x 1010 viable spores/ha.
 The product can be applied to nothern jointvetch that is at least 30 cm tall in
soybean.
 Late afternoon applications are most effective.
 This mycoherbicide can be mixed with the herbicide aciflurofen, but not with
fungicide, insecticide, or other herbicides.

Identifying Nothern Jointvetch:

Three broadleaf weeds, similar in appearance and belonging to the legume family,
commonly infest rice fields:
 Hemp sesbania or coffeebean.
 Indian jointvetch .
 Northern jointvetch .

Use and Application:

 Collego should be applied to emerged northern jointvetch plants that are
8 to 24 inches tall and have not reached the bloom stage.
 Application of Collego when the leaves of the northern jointvetch are
moist.
 For the highest infection rates relative humidities should be above 80%, and
temperatures around 240C.
 Collego should be applied in the evening, but it also may be applied during early
morning hours while the leaves are still wet with dew.
 Soybean fields should be irrigated just prior to application; rice fields should be
flooded.
 Fungicides will reduce activity of Collego and should not be applied 21 days
before or after Collego application.

Table 1. Control of nothern jointvetch in rice fields with C.g.a.

 Area treated Average
Year Tests (No)
(ha) control (%)
1972 1 1 99
1973 4 32 97
1974 8 191 76
1975 17 254 94
1976 20 172 93
1977 19 218 98
1978 13 112 94
1979 17 157 96
1980 22 214 86
1981 15 248 98
1982 2 18 90
US Dept. (Arkansas University) Smith et al. (1986)
Smith et al. (1986) taken [Link] test in different years. They observed that effective average
control in all years from 76 to 99%.

Table 2. Control of nothern Jointvetch in soybean fields with C.g.a.

Tests Area treated Average

Year
(No) (ha) control (%)
1976 4 15 100
1977 6 48 99
1978 2 20 100
1979 5 39 100
1980 7 67 91
1981 5 58 100
US Dept. (Arkansas University) Smith et al (1986)
Smith et al (1986) observed that 100% average control in most of the years and in
remaining years above 90% average control which is very good.

3) Colletotrichum gloesporiodes f. sp. malvae:-

 BIOMAL is the trade name given to a formulation containing the fungal pathogen
C. gloeosporiodes f. sp. malvae for control of round-leaved mallow in lentil and
flax crops in Canada.
 This fungus penetrates the leaf and infects the entire plant by causing the weed rot
and kills it by preventing it from taking up water or nutrients.
 Environmental conditions necessary for infection include 16 to 20 hr of free water
at 200C to 250C. So applications must be made with irrigation.
 The fungus is now being developed for market in 2001 as ‘Mallet W.P.' in both
the U.S.A. and Canada.
 The first symptoms of the disease are dark sunken stem lesions. These turn
grayish in the center with an almost black margin.
  Lesions girdle the stems or petioles resulting in wilting above the lesion. (Smith,
2004).

4) Alternaria destruens (Smolder TM):

 Alternaria destruens a fungal pathogen of dodders (Cuscuta spp.) was discovered
and developed as a bioherbicide by Thomas [Link] (1986, 1987). It causes a
blight disease on several dodder species, and the disease reduces the parasite’s
spread and host damage.
 Alternaria destruens is highly effective in mitigating the damage by dodder
under field conditions. Field trials have been conducted in carrot, cranberry, sunn
hemp, and others. ([Link]).

Facts About SmolderTM:

 The 50% reduction was observed in number of dodder seedlings.
 Does not prevent emergence of dodder but kills dodder before much damage can
be done to the crop.
 Post-emergence Conclusions: Dodder dies after about 14 days after application.

B) Bacteria:
Plant Pathogenic Bacteria:
1) Xanthomonas campestris pv. poannua post-emergence activity on annual bluegrass in

bermudagrass lawns.
2) Pseudomonas syringae pv. tagetis on Canada thistle in soybean.

1) Xanthomonas campestris pv. Poannua:-

 Xanthomonas campestris pv. Poannua is under development as a biotic agent for
control of annual blue grass, a winter annual weed is turf grass.
 This pathogen is applied as a bacterial suspension in liquid to freshly mowed
grass and enters and infects annual blue grass tissues.
 The bacterium spreads through the xylem of infected leaves and causing wilt of
the entire plant and subsequent death.
 Limited field trials revealed that X. campestris pv. Poannua provided up to 91%
control of annual blue grass in bermuda grass turf;however, control was affected
by date and rate of application.

Table 3 Percentage of annual blue grass (Poa annua L.) controlled by Xanthomonas
campestris pv. Poannua or fenarimolb in recreational turf at 10 sites.

Location % control
 Xcpc Fenarimol
Florida 63 13
Florida 50 40
Florida 80 5
Mississippi 58 55
Texas 92 83
Texas 71 84
North Carolina 53 77
South Carolina 75 8
Georgia 86 58
Georgia 75 28
Canada Johnson et al. (1996)
Johnson et al. (1996) reported the percent control of annual blue grass by
Xanthomonas campestris pv. Poannua or fenarimol in different locations they concluded
that, highest percent control was observed with Xanthomonas campestris pv. Poannua at
all locations as compared to fenarimol.

2) Pseudomonas syringae pv. tagetis:

 Pseudomonas syringae pv. tagetis produces “tagetitoxin” that causes apical
chlorosis in many plants of the Compositae.
 The strain under investigation for biological control was isolated from chlorotic
terminal leaves of Canada thistle.
 Cell suspension of this pathogen combined with a surfectant promotes rapid
infection of leaves when applied as a post-emergence spray.
 Disease symptoms include chlorosis and followed by stunting, necrosis and
sometimes death.
 Growth of other composite weeds, including common cocklebur, sunflower, and
common rage weed, is also suppressed by this pathogen.
Canada thistle:
 Colony forming perennial from deep and extensive horizontal roots.
 Leaves lance shaped with spiny tips.

Table 4 Incidence of apical chlorosis in host seedlings 14d after spray inoculation
with pseudomonas syringe pv. tagetis (5 X 108 cfub/ml) in apex suspension
with or without surfactant.

Host Percentage seedlings infected

 Without surfactant With surfactant
Canada thistle 0 100
Common ragweed 5 100
Sunflower 1 100
Marigold 3 100
Canada Johnson et al (1996)
Johnson et al (1996) observed that 100% seedling infected with surfactant as
compared to without surfactant which is very low of all weeds.

C) Virus:
Bioherbicide Agent: Tobacco mild green mosaic virus
Scientific name: Tobacco mild green mosaic tobamovirus.
 It is a well-known, well-studied, plant virus in the Tobamovirus genus.
 Primarily a pathogen of plants in the Solanaceae.
 Strictly a plant pathogen; not a threat to humans, animals, or insects.
 We are using a naturally occurring isolate of TMGMV found in Florida.
 Not genetically modified in any way.

Tropical soda apple (TSA), “The Plant From Hell”:

 A member of the Solanaceae.
 Noxious weed (under both federal and state designations).
 Originally from South America.
 Invasive weed in Florida and seven south eastern states and spreading.
 Primarily a problem in pastures, but also in sod production, citrus, and natural
areas.

Effects of TMGMV on TSA:

 TSA plants of all ages from the seedling stage to full maturity are highly
susceptible to this virus.
 Inoculated plants may develop foliar local lesions.
 About three weeks after inoculation in TSA plants observed wilting.
 Typically the wilted plants die completely without regrowth.

Table 5. Effective control of tropical soda apple in pastures with tobacco mild green
mosaic virus.

Application method Test site located in % TSA kill

Hand-inoculation Hendry County 67.0
 Highlands County 63.0
Osceola Country 1 59.7
Osceola Country 2 88.4
Leaf-infiltration,20 psi
Alachua Country 1 93.0
Alachua Country 2 97.0
Hendry County 35.0
Foliar spray, 20 psi
Highlands County 40.0
Florida (Charudattan, 2002)
Charudattan, (2002) concluded that the highest percent kill of tropical soda apple
by using tobacco mild green mosaic virus with leaf-infiltration method as compared to
hand-inoculation and foliar spray.

Chemical – Bioherbicidal Studies:

 Synergism:
Cooperative action of two (or more) agents (chemicals, bioherbicides, or
combinations) such that the observed response of a target organism (weed) to
their combined application appears to be greater than the predicted effect based on
the expected response to each component applied separately.
 Antagonism:
Combined action of two agents (chemicals, bioherbicides, or
combinations) such that the observed response of their combination appears to be
less than predicted.
 Additive effect:
Cooperative action of two (or more) agents (chemicals, bioherbicides, or
combinations) resulting in an observed response equal to the sum of that predicted
by each independently (Hoagland, 1996).

Limitations of Bioherbicides:
 Restricted commercial potential.
 Dew period and temperature regime for disease development.
 Formulation, shelf life, delivery system.
 Mixtures with other herbicides, insecticides, fungicides.
 Multiple applications may be necessary to affect crop yield.

Bioherbicides can be improved by

 Virulence: - The degree of pathogenesity of a given pathogen.
 Short Residual.
 Broad host pathogens.
 Host-specific pathogen.

Constraints on development of bioherbicide:

(1) There must be sufficient customer demand and market size.
(2) There should be a cost effective manufacturing process which leads to a stable,
effective and easy to use formulation.
 (3) Bioherbicide development requires a comprehensive understanding of the
pathogen and target weed.
(4) Bioherbicides may be more difficult to apply in the field, so that, the delivery
techniques will also be considered.
(5) The formulated product should be highly efficacious, competitive and safe.
(6) The formulated product should have adequate shelf-life for storage point of view.
(7) There should be sufficient sales revenue.
(8) Ability to again patent protection and registration.

Conclusion:
Biological weed control with microbial agents, often referred to as bioherbicides,
has recently received renewed interest because it is provides an additional weed
management strategy for eradication, suppression and environmentally compatible
method of weed control without residue and pollution problems. Bioherbicides such as
Collego, Devine, and Biomal offers similar advantages that of chemical herbicides have
over mechanical control methods, including selectivity and reduced the erosion risk.

References:
Charudattan, R. (2001). Biocontrol. 46: 229-260.
Charudattan, R. (2002). Fla. Weed Sci. Soc., 25.
Gupta, O.P. (2001). Modern weed management., pp.109-119.
Hoagland (1996). Weed Technology. 10: 651-674.
Johnson, D.R.; Wyse, D.L. and Jones, K. J. (1996). Weed Technol., 10: 621-624.
Smith, R. J. (1986). Weed Sci., 34: 17-23.
Smith, A.E. (2004). Handbook of weed management systems., pp. 311-319.

Websites:
[Link]
[Link]
[Link]