Agricultural Microbiology

Unit 1

Biofertilizer

B.Sc Agriculture II

N2-FIXING BACTERIA AS MICROBIALBIOFERTILIZERS

Atmospheric N2 composes approximately 80 percent of theair we breathe. Although abundant and ubiquitous in theair, N is the most limiting nutrient to plant growth becausethe atmospheric N is not available for plant uptake.

Some bacteria are capable of N2 fixation from theatmospheric N pool. These bacteria form variousassociations with plants:

1. Many free-living N2-fixing bacteria occur in soil.

2. Some have adapted to form symbioses; others haveintimate endophytic associations with plants.

3. Others live in close association in the plant root zone(rhizosphere) without forming intimate endophyticsymbioses.

Rhizobia

The best known and most exploited symbiotic N2-fixing bacteria are those belonging to the familyRhizobiaceae (Rhizobia) and include the followinggenera: Rhizobium, Bradyrhizobium, Sinorhizobium,Azorhizobium, Mesorhizobium, and Allorhizobium(Vance, 1998; Graham andVance, 2000).

More comprehensive information on rhizobialsymbiosis can be found in Spaink et al. (1998).

These bacteria infect legumes and have a globaldistribution ranging from high latitudes in Europe andNorth America to the equator, to tropics in Australiaand South America. In equatorial and tropical areas,legumes are particularly important; they are utilized insylvopastoral and agroforestry systems

Legume inoculation is an old practice that has beencarried out for more than a century in agriculturalsystems in the United States and the United Kingdom.

Inoculation is particularly important when local andresident soil rhizobial populations are either absent orknown to be very low.

For example, acidic soils generally contain no or lowpopulation densities of the alfalfa rhizobial symbiontSinorhizobium meliloti, whereas basic soils contain alow inoculum potential of Bradyrhizobium sp., arhizobial symbiont of Lupinus spp.

Under such conditions inoculation with compatiblerhizobia is likely to prove highly advantageous.

The rhizobial inoculum can be produced and appliedin numerous ways.

Inoculum can be prepared as powder, liquid, andgranular formulations.

Granular formulations are convenient as they allowcontrol of placement and application rate.

An additional important feature of an inoculum is theselection of the carrier (e.g., peat, perlite, mineral soil,charcoal). Whichever the carrier, its sterilization isnecessary to maximize inoculum survival andsubsequent infection rate

Frankia Frankia is the genus of N2-fixing actinomycetes that arecapable of infecting and nodulating a group of eightfamilies of mainly woody plants.

These socalled actinorhizal plants are used in landreclamation, for timber and fuel wood production, inmixed plantations, for windbreaks, as well as forshelterbelts along deserts and coastlines.

Actinorhizal Hippophaë rhamnoides is cultivated for itsberries. Frankia N2 fixation has been estimated to besimilar to rhizobial symbioses.

Despite the potential importance of Frankia symbiosis,only limited information is available for inoculationpractices and their use.

Frankia inoculation can be advantageous in aridenvironments, disturbed sites, and areas where nativeactinorhizal plants are absent

CYANOBACTERIA Cyanobacteria are ecologically important. For example, an

aquatic cyanobacterium, Trichodesmium, contributesapproximately 36 percent of global N2 fixation.

Cyanobacterial N2 fixation has been essential in thecultivation of rice. Until the end of the 1970s Azolla-Anabaena symbiosis was the major N source for the 6.5 106ha of rice cultivation in China.

Presently, population pressure and increased labor costshave decreased the reliance on cyanobacterial symbioses inChinese rice cultivation.

In Uruguay and many parts of Asia, the cyanobacteria stillhave a vital importance for rice-field fertility.

Cyanobacteria and their N2 fixation find furtherapplication in the remediation of arid soils. In the Saheliansoil in Niger, N2-fixing cyanobacteria are present in the soilsurface crust.

These cyanobacteria have been shown to increase soil Ncontent , therefore bearing a great promise for reclamationof extreme, arid environments.

N2-FIXING ASSOCIATED BACTERIA

In addition to symbiotic bacteria infecting plant roots,numerous taxa of less intimately associated N2-fixingbacteria can be considered for crop yield improvement.

Examples of such bacteria include Acetobacterdiazotrophicus and Herbaspirillum spp. associated withsugarcane, sorghum, and maize, Azoarcus spp. associatedwith kallar grass (Leptochloa fusca), and Alcaligenes,Azospirillum, Bacillus, Enterobacter, Herbaspirillum,Klebsiella, Pseudomonas, and Rhizobium associated withrice and maize.

The genus Azospirillum colonizes a great variety of annualand perennial plants, many of which have never beenreported to be colonized by N2-fixing bacteria.

Accordingly, Azospirillum possesses a great potential as ageneral root colonizer, whose use is not limited by hostspecificity

Indeed, several studies indicate that Azospirillum canincrease the growth of various crops. These includesunflower, carrot, oak, sugarbeet, tomato, eggplant,pepper, and cotton in addition to wheat and rice.

In two decades of field experiments, general consensusis that in 60 to 70 percent of the cases Azospirillumapplication results in a significant crop yield increase .

The yield increases can be substantial, up to 30percent, but generally range from 5 to 30 percent.

These yield increases by Azospirillum are possibly aresult of the production of growth-promotingsubstances rather than N2 fixation

FREE-LIVING N2-FIXING BACTERIA Many free-living bacteria also fix atmospheric N2.

Examples of such free-living bacteria include Azotobacter,Beijerinckia, and Clostridium.

Furthermore, if environmental conditions allow,nodulating bacterial symbionts (e.g., Frankia) of plantroots can also fix N2 when not in a symbiotic association(free-living culture with their plant host.

More interestingly, it has been found that Frankia canoccur and possibly fixes atmospheric N2 in the rhizosphereof nonhost plants. Frankia has been recorded in therhizosphere of Betula pendula and in soil whereactinorhizal plants were not present.

These observations strongly suggest Frankia N2 fixation inthe rhizosphere of nonactinorhizal plants.

These results open exciting possibilities for utilization ofFrankia in biofertilizer applications but should beconfirmed and further evaluated.

PHOSPHORUS-SOLUBILIZING BACTERIA

We first discussed the importance of the bacterial N2fixation because N is generally the most limitingnutrient for plant growth.

Phosphorus is the second most limiting plant nutrientafter N. Total P content in soil is usually high, but mostof this soil P pool is not in forms available for plantuptake. Bacteria that can mobilize P from unavailablesoil pools and increase P availability to plants are ofgreat importance.

Most predominant phosphorus-solubilizing bacteria(PSB) belong to the genera Bacillus and Pseudomonas

Field experiments highlight the potential importanceof PSB. Sundara et al. (2002) applied rock phosphatewith a PSB (Bacillus megaterium var. phosphaticum) inlignite-based culture medium in a field experiment.

They found that without P application PSBamendment could increase sugarcane yield by 12.6percent.

PSB and P fertilizer together reduced the Prequirement by 25 percent. Furthermore, 50 percent ofthe costly superphosphate could be replaced withinexpensive rock phosphate. PSB also improved thesugar yield and juice quality.

In conclusion, PSB may be of greatest value in allowinguse of cheaper P sources (e.g., rock phosphate insteadof superphosphate).

Mycorrhizal Fungi

Benefits of Mycorrhizal Symbiosis to Plant Growth It isnot our intention to provide an extensive reviewon thebenefits of the mycorrhizal symbioses; we will brieflysummarize the available literature with specialemphasis on the crop yield improvement and potentialapplications that may prove useful in designingbiofertilizers.

For more in-depth review, we refer the reader toextensive published volumes on the importance andbenefits of mycorrhizal symbiosis to plant growth andperformance

Mycorrhizal fungi form mutualistic symbioses with a vastmajority of land plants (Smith and Read, 1997) Possiblymore than 80 percent of all land plants form mycorrhizalsymbioses.

The extent of mycorrhizal symbioses emphasizes theancient evolutionary history and potential importance offungal symbioses for plant production and physiology.

The association between plants and their root-colonizingmycorrhizal fungi is a functional symbiosis in which themycorrhizal fungus is obligately or facultatively dependenton host photosynthates and energy.

The plant-acquired carbon is traded for variousmycorrhizal benefits to the host plant. The fungalmycelium that extends from the root surfaces into the soilmatrix captures nutrients from soil solution.

The minuscule diameter of the fungal hyphae increases thesurface area that the plants are able to utilize for theirnutrient acquisition.

Resulting from the more efficient nutrient uptake, plantgrowth is generally improved when mycorrhizal fungicolonize the root systems.

USE OF ARBUSCULAR MYCORRHIZAL FUNGI ASBIOFERTILIZERS

The unculturability and obligate biotrophy ofarbuscular mycorrhizal (AM) fungi have precluded thedevelopment of large-scale inoculation programs.

The only feasible means for production of infectivepropagules is growing the inoculum in symbiosis withliving host plants or in root organ cultures, in otherwords, never in the absence of living host tissue.

Although such production systems provide anadvantage by allowing a continuous monitoring of theinfective capability of the inoculum, their majordrawbacks include extensive production costs, slowturnover time, and difficulty excluding secondary rootcolonizers such as root pathogens.

USE OF ECTOMYCORRHIZAL FUNGI ASBIOFERTILIZERS

The importance of ectomycorrhizal (EM) symbiosis fortree growth and nutrient acquisition was alreadyhypothesized by Frank (1885).

Accordingly, there is a long history of techniquedevelopment for incorporation of EM fungalinoculation into nursery or forestry plantationpractices.

General evidence suggests that tree plantations willfail unless endemic or inoculated EM fungi areavailable on site.

Selection of the inoculated EM fungi has been largelybased on tree crop enhancement and/or applicabilityfor a large-scale inoculum practices.

MULTIPLE INOCULATIONS AND INTERACTIONSAMONG POTENTIAL BIOFERTILIZERS

We refer to the practice of inoculation and introduction ofmore than one fungus and/or bacterium into the targetcrops as multiple inoculation.

The rhizosphere presents a challenging environment tomanipulate in agricultural practices. Plant roots and theircarbohydrates control the bacterial and fungal populationsand their dynamics in the soil matrix adjacent to the plantroots.

Such host-plant and root-microbe interactions may partlyexplain the species-specific responses to inoculationprograms.

The complexity of the rhizosphere environment is furtheremphasized by the various interactions among thedifferent bacteria and fungi possibly competing forresources or facilitating presence and occurrence bymodification of the rhizosphere environment.

For the past five decades humans have almost been whollydependent upon synthetic/organic insecticides. Agricultur e hasbeen revolutionized by the use of chemicals for crop protection,which started in the last 1800 with the introduction of arsenicalinsecticides and Bordeau mixtures as grape fungicide, andprogressing to the very sophisticated compounds available now .

Today, fewer people produce more food at less cost than everbefore. The effect of synthetic chemicals on agriculture has beenso dramatic that conventional agriculture now means us ingchemicals.

Despite the immense benefits, they are used in increasingquantities designed to kill living organisms. However, the veryproperties that give these chemicals useful-long residual actionand high toxicity for a wide spectrum of organisms, have givenrise to serious environmental problems.

Furthermore, the emergence and spread of increasing resistancein many vector species, concerns over environmental pollution,and the ever increasing cost of the new chemical insecticides,make it apparent that vector and pest control can no longer besafely based upon the use of chemicals alone.

Consequently, increasing attention has been directedtoward natural enemies such as predators, parasites,and pathogens.

Unfortunately, none of the predators or parasites canbe mass produced and stored for long periods of time,since they all must be raised in vivo.

It has become evident that there is an urgent need for abiological agent, possessing the desirable propertiesof a chemical pesticide making it highly toxic to thetarget organism, which can be mass produced on anindustrial scale, has a long shelf life and can be safelytransported.

In the mid seventies, WHO and other internationalorganizations initiated studies into existing biologicalcontrol agents and the development of new ones.

Today, biological control is widely regarded as adesirable technique for controlling insects, due to itsminimal environmental impact and its avoidance ofproblems of resistance in the vectors and agriculturalpests.

Of the nearly one million known species of insects,about 15,000 species are considered pests and about300 require some form of control.

Fortunately, most insect pests have pathogenicmicroorganisms associated with them.

Entomopathogens have been suggested as controllingagents of insect pests for over a century, and belong tospecies of fungi , viruses, bacteria, and protozoa.

Insect pathology per se probably had its beginning inthe nineteenth century under the stimulus of Bassiand Pasteur. A significant contribution to microbialcontrol of insects was made by Mechnikoff in 1879and Krassiln ikow in 1888, who were the first todocument that an entomopathogen, a muscordinefungus, Metarrhizium anisopliae could be massproduced and applied as a microbial insecticide tocontrol the grain and the sugar beet pests.

The control of insect pests with bacteria was probablyfirst attempted by d'Herelle in 1914, approximately 35years after Pasteur's description of silkworm diseases.

Apparently the control was not consistent andtherefore interest in bacterial pathogens was curtailed.

However, after a lag period of nearly 30 years, White andDutky succeeded in 1940 in demonstrating a control of theJapanese beetle by distributing spores of the milky diseasebacterium Bacillus popilliae.

This success stimulated further investigations of bacteriaand literature began appearing on the effectiveness ofBacillus thuringiensis.

The issuing of eight patents between 1960 and 1963 forB.thuringiensis led to a revived interest in bacterialinsecticides. The use of viruses to control insect pests wasstimulated by the studies of Balch and Bird in 1944 andSteinhaus and Thompson in 1949, respectively.

This initial interest is presently having a rebirth, as isevidenced by the recent registration of the first viralpesticide in the United States by the EnvironmentalProtection Agency (EPA)

Of these, bacteria, viruses and some fungi , because oftheir known effectiveness and relative lack of toxicityor pathogenicity to nontarget animals and plants, havebeen developed into commercial products.

Biological control is generally man's use of a speciallychosen living organism to control a particular pest.

This chosen organism might be a predator, parasite orinfectious disease, which attack the harmful insects.

Biological control methods can be used as part of anoverall integrated pest management program toreduce the legal, environmental, and public safetyhazards of chemicals. In addition, it may be a moreeconomical alternative to some insecticides.

Some biological control measures can actually preventeconomic damage to agricultural crops. Unlike mostinsecticides, biological controls are often very specificfor a particular pest.

There is less danger of impact on the environment andwater quality and they offer a more environmentallyfriendly alternative to chemical insecticides.

They could also be used where pests have developedresistance to conventional pesticides.

Unfortunately, research and development of biologicalinsecticides attracts very little financial supportcompared to that given toward the discovery ofchemical pesticides.

It is becoming clear that more attention needs to be givento the selection of broad spectrum biopesticides andimprovements in the production, formulation andapplication technologies.

Efforts need to be made to optimize the impact of theseagents by integrating them with other novel cropprotection strategies.

Successful use of biological control requires a greaterunderstanding of the biology of both the pest and itsenemies.

In some cases, biological control may be more costlycompared to the use of pesticides. Often the results ofusing biological control are not as dramatic or quick as it iswith chemical pesticides.

Most natural enemies attack only specific types of insectsunlike broad-spectrum insecticides which may kill a widerange of insects.

Today biological control is regarded as a desirabletechnique for controlling insects, due to its minimalenvironmental impact and preventing thedevelopment of resistance in vectors.

Specific biotoxin-producing strains of Bacillusthuringiensis var. israelensis or B.sphaericus have beenused throughout the world to suppress or eliminatethe larval stages of mosquitoes, particularly wheremalaria, filariasis or certain arboviruses are present.

Bacillus thuringiensis var. israelensis is also effectiveagainst the larval stages of Simulium spp., vectors ofriver blindness in man (onchocerciasis) in tropicalAfrica, and the cause of severe ‘fly worry’ in domesticlivestock in several regions of the world.

Depending on the specific control programmed,chemical larvicide's may precede or alternate with theuse of Bacillus.

Host treatment for onchocerciasis or filariaisis mayalso be performed. Studies conducted to date haveshown no significant effect of these bacteria and theirtoxins on vertebrates and only minimal effects onsome non-target arthropods and crustaceans.

Development of resistance is apparently less of aproblem than with chemical pesticides.

These and other potential problems are continuouslybeing monitored and investigated.

Other potential tools which could be used in the future forarea-wide biological control programme against insectvectors/pests of veterinary importance include species-specific sex pheromones.

These are presently being used as attractants for trappingand monitoring of insects and for mating disruption. Inaddition, several parasites and pathogens of vector/pestspecies are under continuous investigation and areproviding promising results.

Hopefully, the not too distant future will witness thedevelopment of further biological control techniques ofsufficient scope to free entire regions of pathogenic agentsor vectors which cause or transmit significant diseases notonly of domestic livestock and humans, but also of free-living wildlife.

Virtually all pest populations are affected by naturalenemies to some extent.

In many cases, natural enemies are the primary regulating forceof the pest populations

. Natural controls include effects of natural enemies (predators,parasites, pathogens), other biotic (living) factors such as foodavailability and competition, and abiotic (non-living) factorssuch as weather and soil.

In pest management, biological control usually refers to theaction of parasites, predators or pathogens, on a pest populationwhich reduces its numbers below a level causing economicinjury.

Herbivorous insects and pathogens that attack pest weeds arealso considered bio- control agents. Biological control is a part ofnatural control and can apply to any type of organism, pest ornot, and regardless of whether the bio -control agent occursnaturally, is introduced by humans, or manipulated in any way.

Biological control differs from chemical, cultural, andmechanical controls in that it requires maintenance of somelevel of food supply (e.g., pest) in order for the biocontrol agentto survive and flourish.

Therefore, biological control alone is not a means bywhich to obtain pest eradication. Biological control isdefined as the action of natural enemies.

It can be divided into 2 broad categories, natural biologicalcontrol and applied biological control. Natural biologicalcontrol occurs where native or co-evolved natural enemiesreduce native arthropod populations, whereas appliedbiological control involves human intervention to enhancenatural enemy activities.

Applied biological control can be further separated into

(a) classical biological control, where exotic naturalenemies are introduced against an exotic or native pest, or

(b) augmentative biological control, where humanintervention occurs to enhance the effectiveness of thenatural enemies already present in an area throughmanipulation of the environment.

Numerous species of plant-feeding insects have been evaluatedfor control of pest weeds. The greatest successes have been inrangelands, forests, and other natural habitats where other weedcontrol approaches (e.g., herbicides, cultivation) are impracticalor uneconomical.

Some pathogens have also been looked at as weed biocontrolagents (e.g., plant rusts). The goal, while using the weedbiocontrol agents, is generally to reduce the weed populationand not to eradicate it.

Importation of a biocontrol agent from the region of origin ofthe weed has been the most common approach. It is generally along-term process which requires sustained efforts, but whichcan reap long-term benefits.

A few of bacteria are highly effective at killing insects. The mostimportant of these is Bacillus thuringiensis (Bt). It occursnaturally in insect-rich locations, including soil, plant surfacesand grain stores.

It kills a range of insect orders and is the most widely usedmicrobial biopesticide. It is also used in transgenic crops.

There are over 40 Bt products available worldwide for control ofcaterpillars, beetles and blood-feeding flies such as mosquitoes.Together, these account for 1% of the world insecticide market.

As part of its life cycle, Bt produces protein crystalswhich have insecticidal properties.

When ingested, the crystals paralyze the digestivetracts of insects, often killing them within 24-48hours.

Different Bt strains produce crystals with slightlydifferent properties, and the crystals from each strainare specific for a small number of related insectspecies.

Over 1600 viruses have been recorded from more thana thousand species of insects. A family of viruses calledbaculoviruses is the most popular choice for microbialcontrol as they are distinct from any type of virusrecorded from vertebrates.

They have been used regularly for pest control sincethe 1950s, particularly in forestry where they have beenhighly effective at controlling sawflies.

Baculoviruses are very species, mostly caterpillars andsawflies, but also some species of beetle and flies.Baculoviruses infect their hosts through ingestion.

Virus particles invade the cells of the gut beforecolonizing the rest of the body.

Infection reduces mobility and feeding and insects arekilled in five to eight days. Mass production ofbaculoviruses can be done only in insects, but this iseconomically viable for larger hosts such ascaterpillars, and formulation and application arestraightforward.

At present, there are approximately 16 productsavailable for use, or under development, mostly forcontrol of caterpillar pests.

Commercial products are available in Switzerland,Germany and Spain for the control of codling mothand the summer fruit tortrix.

Products are also available in the USA for the controlof tobacco bollworms on vegetables, ornamentals,tomatoes and cotton.

Over 750 species of fungi kill insects.Entomopathogenic fungi invade their hosts usingspores that grow through the cuticle, and hence theyare particularly suited for control of pests withpiercing mouthparts, such as aphids and whiteflies,which are unlikely to acquire pathogens through feeding.

Infection requires high humidity at the insect surface,but this can be overcome using oil-based formulations.

About 20 products are available worldwide formanaging sap-feeding insects, beetles, caterpillars,flies and locusts.

In the USA, and some countries in Europe, productsbased on the fungus Beauveria bassiana are becomingavailable for the control of a range of glasshouse pests.

Entomopathogenic nematode worms are just visible tothe naked eye, being about 0.5 mm in length. Juvenilenematodes parasitize their hosts by directlypenetrating the cuticle of through natural openings.

They then introduce symbiotic bacteria, whichmultiply rapidly and cause death by septicaemia, oftenwithin 48 hours.

The bacteria break down the insect body, whichprovides food for the nematodes.

After the insect has died, the juvenile nematodesdevelop to adults and reproduce.

A new generation of infective juveniles emerges 8-14days after infection.

Unlike other entomopathogens, nematodes are exemptfrom registration and so have been popular choices forcommercialization.

Over 60 products are available in Europe.

Nematodes require moist conditions to operate andhave been marketed predominantly against soil pests,such as vine weevil and sciarid fly larvae.

However they may also control foliar pests, forexample Nemasys (Becker Underwood) which can beused to control western flower thrips.

Like other natural enemies, nematodes are affected byenvironmental conditions.

Protozoan diseases of insects are ubiquitous and comprisean important regulatory role in insect populations. Theyare generally host specific and slow acting, most oftenproducing chronic infections.

The biologies of most entomopathogenic protozoa arecomplex. They develop only in living hosts and manyspecies require an intermediate host.

Species in the Microsporida are among the mostcommonly observed. Their main advantages are persistenceand recycling in host populations and their debilitatingeffect on reproduction and overall fitness of target insects.

As inundatively applied microbial control agents, only afew species have been moderately successful. Thegrasshopper pathogen Nosema locustae Canning is theonly species that has been registered and commerciallydeveloped.

The main disadvantages of the protozoa as inundativelyapplied microbial control agents are the requirement for invivo production and low levels on immediate mortality.

Microbial insect control utilizes pathogenicmicroorganisms isolated from diseased insects duringnaturally occurring epidemics.

Typically, such epidemics only occur when pestpopulation densities are high and usually afterappreciable damage have been done to crops.

Over 400 species of fungi and more than 90 species ofbacteria which infect insects have been describedincluding Bacillus thuringiensis , varieties of which aremanufactured and sold throughout the worldprimarily for the control of caterpillar pests and morerecently mosquitoes and black flies.

Among fungal pesticides, five have been introducedsince 1979, and three in 1981. Many countries withcentrally planned economies have been using fungalpesticides successfully for many years.

Sofar, more than 40,000 species of Bacillusthuringiensis have been isolated and identified as belonging to 39 serotypes.

These organisms are active against either Lepidoptera ,or Diptera or Coleoptera.

Bacillus Thuringiensis Maximizing the potential forsuccessfully developing and deploying a biocontrolproduct begins with a carefully crafted microbial screeningprocedure, proceeds with developing mass productionprotocols that optimize product quantity and quality, andends with devising a product formulation that preservesshelf-life, aids product delivery, and enhances bioactivity.

Microbial selection procedures that require prospectivebiocontrol agents to possess both efficacy and amenabilityto production in liquid culture increase the likelihood ofselecting agents with enhanced commercial developmentpotential.

Scale-up of biomass production procedures must optimizeproduct quantity without compromise of product efficacyor amenability to stabilization and formulation.

Formulation of Bacillus spp. for use against plantpathogens is an enormous topic in general terms butlimited in published specifics regarding formulationsused in commercially available products.

Types of formulations include dry products such aswettable powders, dusts, and granules, and liquidproducts including cell suspensions in water, oils, andemulsions.

Cells can also be microencapsulated. Considerationscritical to designing successful formulations ofmicrobial biomass are many fold and includepreserving biomass viability during stabilization,drying, and rehydration; aiding biomass delivery,target coverage, and target adhesion; and enhancingbiomass survival and efficacy after delivery to thetarget.

Over 90 species of naturally occurring, insect specific(Entomopathogenic) bacteria have been isolated frominsects, plants and the soil, but only a few have beenstudied intensively.

Much attention has been given to Bacillusthuringiensis , a species that has been developed as acommercial microbial insecticide since 1960 and issold under various trade names.

Bacillus thuringiensis (Bt) occurs naturally in the soiland on plants.

Different varieties of this bacterium produce a crystalprotein that is toxic to specific groups of insects.

The entomopathogenic microorganism Bacillusthuringiensis is one of the most promising biologicalcontrol agents for pest and insect management sincemany strains are toxic specifically for Lepidopteranand strain Bacillus thuringiensis serotype H-14 ishighly toxic to Dipterans.

The first data on the existence and activity of the newstrain B. thuringiensis H-14 appeared in 1977.

This bacterium was first recorded in 1901 as the causeof the damaging "Sotto" disease in silkworms in Japan.

It was again isolated in 1927 by Maltes in Germany andgiven the name B.thuringiensis.

Bacillus thuringiensis (B.t.) is a gram positivebacterium characterized by a parasporal crystallineprotein inclusion (Figure 1). The proteins are highlytoxic to pests and specific in their activity over the past40 years.

The commercial use of B.thuringiensis as a pesticidehas been largely restricted to a narrow range ofLepidopteran (Caterpillar) pests. In recent years,however, investigators have discovered B.t. pesticideswith specificities over a much broader range of pests.

The toxin genes have been isolated and sequenced,and recombinant DNA-based B.t. products producedand approved. Many of the newly discovered strainshave activities that would extend the use of Bacillusthuringiensis beyond traditional agricultural markets.

Bacillus thuringiensis products account for 90-95percent of the total biopesticide market which hasgrown from $ 24 M in 1980 to $ 107 M in 1989, and isforecast to expand at an annual rate of 11 percent.

The availability of a large number of diverse B.t. toxinsmay also enable farmers to adopt usage strategies thatminimize the risk of B.t. resistant pests.

The B.t. strains known are classified according to theirH antigens into 27 groups and 7 subgroups andaccording to structure and molecular organization ofthe genes coding for the parasporal delta-endotoxins.

Only a few strains are used commercially as controlagents. The main bacteria are different varieties ofBacillus thuringiensis, Bacillus sphaericus and Bacilluspopilliae

.

Mode of Action

Numerous moth and butterfly larvae and some beetleand fly larvae are susceptible to infection.Formulations of Bt variety kurstaki are available for thecontrol of many caterpillar pests including importedcabbageworm, cabbage looper, hornworms, Europeancorn borer, cutworms, some armyworms, diamond-back moth, spruce budworm, bagworms, tentcaterpillars, gypsy moth caterpillars and other forestcaterpillars, and Indianmeal moth larvae in storedgrain.

Less well controlled are corn earworms on corn,codling moth, peach tree and squash vine borers.

Formulations of Bt variety tenebrionis and varietysan diego have been registered for the use against theColorado potato beetle larvae and elm leaf beetleadults and larvae. Bt Variety israelensis is marketed foruse against black flies and mosquitoes, fungus gnats.Unless used on a community-wide basis, it is probablymore effective to eliminate standing water and controlweeds at the edges of ponds.

Bt variety aizawai is used to control wax moth larvae inbee hives and various caterpillars. It is important forthe control of diamondback moth caterpillar whichhas developed resistance to Bt variety kurstaki in someareas.

The toxic crystal Bt protein in commercialformulations is only effective when eaten by insectswith a specific (usually alkaline) gut pH and thespecific gut membrane structures required to bind thetoxin.

Not only must the insect have the correct physiologyand be at a susceptible stage of development, but thebacterium must be eaten in sufficient quantity.

When ingested by a susceptible insect, the proteintoxin damages the gut lining, leading to gut paralysis.The affected insects stop feeding and die from thecombined effects of starvation and tissue damage.

Bt spores do not usually spread to other insects orcause disease outbreaks on their own as occurs withmany pathogens.

Bacillus Thuringiensis Var.kurstaki In 1970, Dulmageisolated the HD-1 strain of Bt var. kurstaki and itbecame commercially available shortly thereafter. It isused today for the production of most Bt var kurstakiformulations used to control defoliating forestLepidoptera in North America.

The HD-1 strain is a serotype 3a 3b, and the crystal hasa fairly broad spectrum of activity against a largenumber of Lepidoptera .

Four companies produce various types of formulations(e.g., a queous flowable suspension, nonaqueousemulsifiable suspension, oil flowable) of the HD-1strain for use against gypsy moth.

Each formulation contains inert ingredients which areunique and various additives (e.g., stickers) can beincluded to produce the final mix.

Loss of residual toxicity of Bt var kurstaki on foliagecan result from degradation by sunlight, leaftemperatures, drying, washing off by rain, microbialdegradation, and leaf chemistry.

Solar radiation appears to be the key factor affectingthe survival of Bt var kurstaki spores and crystalsdeposited on foliage. In a series of Bt var kurstakibioassays, the half-life of its insecticidal activity forearly stage gypsy moth larvae in the field has beenestimated at 12-32 hours.

In spite of this short half-life, a deposition of 75 IU cm-2 from a 90 BIU ha -1 application will give, on theaverage, insecticidal activity against early stage gypsymoth larvae of at least an LC 50 for 4 to 6 days.

Many safety tests have been performed with Bt varkurstaki . None of the vertebrates tested showed anyabnormal reaction in terms of external symptoms orinternal pathologies.

Nevertheless, vertebrate species that rely onlepidopterans as a food source (e.g., Virginia Big-earedbat, insectivorous birds) have the potential of beingindirectly affected by the Bt. Var kurstaki suppressionprograms.

For example, Rodenhouse and Holmes showed that areduction in biomass of lepidopteran larvae followingBt. Var kurstaki application led to significantly fewernesting attempts of certain birds.

Many lepidoptera that co-occur with a pest species arealso susceptible to Bt. Var kurstaki . Of particularconcern would be non-target impacts on lepidopteransthat are important as pollinators, in the suppression ofweedy plants, and other ecosystem functions.

For example, James and coworkers showed in 1992 thatBt. Var kurstaki is toxic to late, but not early, instarlarvae of the cinnabar moth (Tyria jacobaeae), which isan important species in the control of the noxiousweed, tansy ragwort.

Impacts on rare and endangered species are also ofspecial concern.

Bacillus thuringiensis var kurstaki is a bacterium thatoffers natural biodegradable, safe control of pests, andcan be sprayed onto plan ts, where it is eaten by leaf-eating insects, including loopers, hornworms,earworms, cat erpillars, gypsy moths, oakworms, mealand flour moths, diamondback moths, fruitworms,leaf folders, and most species of leaf-feeding moth andbutterfly larvae.

The Bt var kurstaki disrupts the insects' digestivesystem and they starve. It is specific to certain pestinsects, can be used with nematodes, and iscompletely environmentally safe.

Crops can be harvested the day after any Bt variety isapplied

Bacillus thuringiensis var kurstaki formulations arespecific to lepidopteran larvae which is preferable topreviously used broader spectrum chemicalinsecticides. Technical developments in geneticengine ering and molecular biology are providingopportunities for the development of geneticallymanipulated strains of Bt. Var kurstaki and transfer oftoxin-coding genes into other bacteria (cloning) orplant species.

In the near future , it may be possible to engineer moretaxon specific strains of Bt. Var kurstaki that can bedeveloped and commercially produced.

Bacillus Popilliae and Bacillus Lentimorbus Thebacteria grouped under the name B. popilliae causemilky diseases of beetles (coleoptera), especially of thebeetle family Scarabaeidae .

In practice, B. popilliae has been used intensively andalmost exclusively for control of the Japanese beetle,Popillia japonica.

The beetle causes serious damage in North America,since the adult beetle feeds on a wide range ofornamental and crop plants , eating the tissuesbetween the reins, and accumulates on ripening fruit,causing substantial damage.

It is also a problem in the larval stage because theadult beetle lay their eggs in turf and the grubs destroythe grass roots.

Beetle larvae killed by Bacillus popilliae andB.lentimorbus may turn white, hence the name "milkydisease"

By the 1930s, the infestation had become so extensivethat a search for a control measure was undertakenwhich led to the discovery in nature of some diseasedlarvae.

Milky spore bacteria were isolated. The term "milkydisease" comes from the larva's pure white appearancewhen infected with B. popilliae. B. popilliae was thefirst insect pathogen to be registered in the U. S. as amicrobial control agent

Bacillus popilliae and Bacillus lentimorbus are related,naturally occurring bacteria that have been mass-produced for the control of Japanese beetle larvae inturf since the 1940s.

Several commercial products are available. Thebacteria, usually applied to the soil, cause "milkydisease".

Milky disease spores may reproduce within the beetlelarvae and establish a resident population capable ofcausing mortality over several seasons if the soil issufficiently warm and moist through the summermonths.

It may take several seasons for the disease to controlthe pest, and it is preferable to treat a broad area toreduce the impact of immigrating healthy beetles.

B. popilliae is a Gram-negative spore-forming rod of 1.3to 2.5 0.5 to 0.8 micrometers. A fastidious organism,B. popilliae can only be cultivated on rich mediacontaining yeast extract, casein hydrolysate, or anequivalent amino acid source, and sugars.

Several amino acids are known to be required forgrowth, as well as the vitamins thiamine andbarbituric acid.

Trehalose, the sugar found in insect hemolymph, is afavored carbon source, although glucose can also beused.

Japanese beetle is the exclusive host of the strain of B.popilliae which is sold commercially.

However, other B. popilliae strains (and B. lentimorbus

, which is considered a strain of B. popilliae by someexperts) have other scarab hosts and are specific todifferent beetles in the family Scarabaeidae , whichincludes the Japanese beetle and the chafers-important Pasture pest s, but also the beneficial dungbeetles.

Spores which reside in the soil and have been ingestedby beetle larvae germinate in the larva's gut within 2days and the vegetative cells proliferate, attainingmaximum numbers within 3 to 5 days.

By this time, some of the cells have penetrated the gutwall and have begun to grow in the hemolymph, wherelarge numbers of cells develop after 5 to 10 days.

High quality milky spore powder, containing thebacterium Bacillus popillae, inoculates an area of turfand can control Japanese beetle grubs (Popilliajaponica ) for decades.

The milky spore bacteria infect and then multiplywithin the grub host. When the larva dies the diseaseis spread to surrounding areas of the lawn or garden.

The spores produced in a host are almost invulnerableto climatic conditions and can spend years in the soilwaiting to infect other grubs.

For best results, the application should be done whenthe soil is warm.

References

Handbook of Microbial Biofertilizers M. K. Rai,