Formulation of Living Biological Control Agents with Alginate - ACS

Chapter 19

Formulation of Living Biological Control Agents with Alginate

Downloaded by OHIO STATE UNIV LIBRARIES on June 7, 2012 | http://pubs.acs.org Publication Date: June 24, 1988 | doi: 10.1021/bk-1988-0371.ch019

William J. Connick, Jr. Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179 Alginate i s a water-soluble polysaccharide gum that has excellent gel-forming properties· Living organisms that are capable of b i o l o g i c a l l y c o n t r o l l i n g a variety of a g r i c u l t u r a l pests can e a s i l y be entrapped i n a calcium alginate matrix by a f a s t , gentle, aqueous, room temperature process. The gel beads, granules, or seed coatings that are produced by the process are biodegrada ble i n the environment. Weed-killing fungi ( A l t e r n a r i a , Fusarium, and P h y l l o s t i c t a spp.), antagonistic fungi (Trichoderma, Gliocladium, Talaromyces, and P e n i c i l l i u m spp.) and bacteria (Pseudomonas and B a c i l l u s spp.) which can control soilborne plant disease pathogens, and i n s e c t - k i l l i n g nematodes (Steinernema and Heterorhabditis spp.) have all been formulated successfully using alginate. This r e l a t i v e l y new use of alginate properties to formulate biocontrol agents i s a v e r s a t i l e and promising development i n a g r i c u l t u r a l research which i s reviewed i n t h i s paper. Alginate i s a l i n e a r , (1-4)-linked copolymer of α-L-guluronate and 3-D-mannuronate that i s a unique, water-soluble polysaccharide gum (1). Most of the alginate of commerce i s extracted from Macrocystis p y r i f e r a , a giant kelp, but i t i s an important s t r u c t u r a l component of a l l Phaeophyceae, the brown seaweeds* Gel-forming, thickening, suspending, film-forming, and s t a b i l i z i n g properties make alginate solutions ideal for many food, pharmaceutical, cosmetic, a g r i c u l t u r a l , and i n d u s t r i a l applications* As an adjuvant for pesticide spray formulations, alginate acts as a suspending and filming agent (1). Viscous solutions of sodium alginate containing the herbicide diquat dibromide have been extruded into water to control submerged aquatic weeds (2-4). Sodium alginate solutions can e a s i l y be gelled by reaction with c e r t a i n metal cations such as calcium by a mechanism that i s known as ionotropic gelation* Alginate gels have been widely used for many years i n food products (1). More recently, they have been used to immobilize microbial c e l l s and enzymes for use i n flow-through This chapter not subject to U.S. copyright Published 1988 American Chemical Society

In Pesticide Formulations; Cross, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


242

PESTICIDE FORMULATIONS: INNOVATIONS AND DEVELOPMENTS

biochemical reactors (5-7) to produce useful chemical compounds such as ethanol, and to immobilize a fungus which decolorizes kraft m i l l effluent (8). In agriculture, the g e l l i n g property of alginate has been u t i l i z e d i n recent years for the preparation of c o n t r o l l e d release formulations of chemical pesticides (9-17). Living organisms such as fungi, bacteria, and nematodes can be readily entrapped i n a non-toxic calcium alginate matrix by a gentle, room temperature, aqueous-based process i n which the organism i s added to a sodium alginate (1-2% by wt.) solution and the resulting mixture i s then added dropwise to a calcium chloride (3% by wt.) solution. Each droplet i s gelled at the surface almost immediately upon immersion i n the calcium chloride (gellant) solution to form a spherical gel bead. A residence time of 1-20 minutes i n the calcium solution allows gelation to proceed from the bead surface toward the center as the Ca ^ ions diffuse inward and react with the carboxylate groups of the alginate. Gel beads containing the organism may be harvested and used i n their "hydrated** state, or dried to form small pellets or granules. Organic or inorganic f i l l e r s , nutrients, or other adjuvants are normally added to the alginate/organism mixture before gelation i n order to improve the f i n a l product. Biodegradable, non-polluting alginate gel beads containing b e n e f i c i a l rhizosphere b a c t e r i a l inoculants (18, 19) have been described. The alginate technique i s also useful for the preparation and storage of large quantities of inoculum for f i e l d evaluation of disease resistance and fungicide evaluation as was done by Boyette and Walker (20) with the soybean-destructive fungus Cercospora k i k u c h i i . B i o l o g i c a l control strategies are v i t a l l y important i n today's agriculture because of the increased number of pests that are resistant to chemical pesticides and because of contamination of surface and ground water supplies by chemical pesticides. This paper reviews the use of alginate gel technology to prepare formulations containing l i v i n g b i o l o g i c a l control agents such as fungi, bacteria, and nematodes for the control of a g r i c u l t u r a l pests such as weeds, soilborne plant pathogens, and insects. +

Weed Control Applications Granular formulations of fungal plant pathogens (mycoherbicides) can be applied preemergence to attack weed seedlings as they emerge from the s o i l when they are the most vulnerable to i n f e c t i o n . Alginate granules containing indigenous weed-killing fungi (Table 1) were f i r s t prepared by Walker and Connick (21). P h y l l o s t i c t a sorgfticola, i s o l a t e d from johnsongrass [Sorghum halepense (L.) Pers.], was included as a representative pycnidium-forming fungus. Each fungus was cultured and formulated separately. The fungi were grown i n a commercial fermentor, the mycelium and growth medium were homogenized, and sodium alginate, water, and k a o l i n f i l l e r were added. Granules that contained the mycelium dispersed throughout were prepared by dropwise addition of the alginate mixture to a calcium chloride solution, and then air-drying the resulting gel beads.


19. CONNICK

Living Biological Control Agents

243

To determine the number of conidia (spores) produced per gram of formulation when sporulation was induced a f t e r 10 weeks storage, granules were placed on moist f i l t e r paper i n p e t r i dishes and were p e r i o d i c a l l y exposed to light from sunlamps. At designated i n t e r v a l s , the conidia were washed from the granules using a d i l u t e surfactant solution and counted under magnification.


The t o t a l number of conidia per gram of sample harvested 2, 5, 7, and 9 days after rewetting was compared to the number of conidia from a single harvest at 9 days (Table I ) .

Table I. Conidia obtained i n single and multiple harvests from alginate granules

Fungus

A l t e r n a r i a cassiae

6

Conidia (no. x 10 /g Formulation)

Multiple Harvest 1.5

A l t e r n a r i a macrospora

2.4

Fusarium

9.6

lateritium

Colletotrichum ma1varum P h y l l o s t i c t a sorghicola

a

a

Single Harvest

11.3 520

The same samples of the granules were rewetted and induced to sporulate after harvest at 2, 5, 7, and 9 days. No conidia were present i n i t i a l l y .

b

A single harvest of conidia was made 9 days a f t e r rewetting the granules. SOURCE: Data from ref. 21. A l l of the fungi that were tested produced new conidia after each harvest during the washing and rewetting cycles to give a b e n e f i c i a l sustained-release effect* Multiple harvests gave higher conidia production for the two Alternaria species than did the single harvest at 9 days. F i e l d observations have confirmed that the pathogens used i n this study, when formulated and packaged as dried granules without conidia, sporulated readily under f i e l d conditions provided that adequate moisture was present. The a b i l i t y of A. cassiae to infect and k i l l sicklepod (Cassia o b t u s i f o l i a L.) seedlings has been demonstrated i n f i e l d plots where the granules were applied preemergence (22).




244

Pycnidium-forming fungi are d i f f i c u l t to produce by conventional techniques, and only a few species have been studied as potential mycoherbicides. In the Walker and Connick (21) study, the P h y l l o s t i c t a species produced numerous pycnidia on the granules. It appears that this method of alginate formulation may be ideal for pycnidium-forming fungi because the conidia need not be removed from the pycnidia for f i e l d application, thus offering better protection for the fungus against adverse environmental conditions. The application of these alginate/fungus granules to s o i l i s their most direct use. However, the granules can also be used as a matrices from which spores can be grown and harvested for other uses such as the formulation of f o l i a r sprays. Boyette and Walker (23) studied the weed control e f f i c a c y of the biocontrol fungus Fusarium l a t e r i t i u m Nees ex F r . formulated as a c o n l d i a l spray or incorporated i n alginate granules. The formulations were applied i n the greenhouse to corn, cotton, and soybeans that were infested with velvetleaf (AbutiIon theophrasti M e d i c ) and p r i c k l y sida (Sida spinosa L . ) , and applied i n the f i e l d to soybeans infested with these same weeds. In the greenhouse, both weeds were e f f e c t i v e l y and equally controlled with the postemergence conldial spray or the preemergence alginate/]^, l a t e r i t i u m granules (Table I I ) . The f i e l d test data i n Table I I also show that the spray and granule applications gave about the same weed control, which was low because of drought conditions that year.

Table I I .

B i o l o g i c a l control of velvetleaf and p r i c k l y sida i n soybeans with Fusarium l a t e r i t i u m i n greenhouse and f i e l d tests

a

Weed Control ( % ) Greenhouse Field Velvetleaf Prickly sida Velvetleaf Prickly sida b

Treatment

c

Conldial spray

91

94

30

36

Alginate/F. l a t e r i t i u m granules

89

92

35

38

a

Controls consisting of surfactant-only spray and granules without fungus gave 0-9% weed control. Weed control was determined by percent stand reduction a f t e r 4 weeks.

b

Spray: postemergence, sprayed to runoff, 1.5 x 10^ conidia/ml; granules: preemergence, 1120 kg/ha rate.

c

Spray: postemergence, applications 1-week apart, 1.5 x 10* conidia/ml; granules: preemergence, 1120 kg/ha rate.

SOURCE: Data from ref. 23.



19. CONNICK

245


The alginate/^, l a t e r i t i u m granules served a dual purpose: (a) they were applied d i r e c t l y to test plots i n a conventional manner; and (b) they were used to grow conidia f o r the spray formulations. In the l a t t e r case, fungus-infested granules were placed i n trays and hydrated with d i s t i l l e d water. The trays were covered with p l a s t i c f i l m and the granules were incubated and exposed p e r i o d i c a l l y to u l t r a v i o l e t l i g h t from sunlamps. The fungus sporulated profusely on the granules and the macroconidia were rinsed from the granules with water. Lindow conducted a f i e l d experiment i n which a c o n i d i a l suspension of an Alternaria species that i s a s p e c i f i c biocontrol agent f o r I t a l i a n t h i s t l e (Carduus pycnocephalus L.) was compared with an alginate granule preparation containing the fungus plus soy flour and cor rime a l (50/50) as nutrients and f i l l e r s (Lindow, S., University of C a l i f o r n i a , Berkeley, personal communication, 1987). The results (Table III) show that the granules, when used with I t a l i a n t h i s t l e seedlings (2-4 cm high) i n the presence of rain splash, were e f f e c t i v e i n delivering viable, infectious propagules to the t h i s t l e s . The infection obtained (judged after 3 weeks) with the granules was better than with the conidial spray formulation. However, i n other t r i a l s where hard rain was not encountered or where the t h i s t l e plants were t a l l e r , i n f e c t i o n was not as severe. This seems to be due to a lack of s u f f i c i e n t dispersal of spores to the target plant from granules that rest on the s o i l surface. Even though the granules are t h i c k l y covered with Alternaria spores, there i s usually not much a i r movement at ground l e v e l to e f f e c t i v e l y disperse the spores. This i s a l i m i t a t i o n inherent i n any granular formulation of a fungus l i k e Alternaria that produces spores which attack the a e r i a l portions of a weed.

Table I I I . Severity of f o l i a r necrosis incited by A l t e r n a r i a sp. on I t a l i a n t h i s t l e (Carduus pycnocephalus). (Lindow, S., personal communication, 1987). a

Treatment

Infection (Percent of l e a f ) ^

Control

0.02

Conidial suspension

0

Alginate/Alternaria granules

a

41.0 50.5

The fungus was applied to 2-4 cm t a l l plants i n a f i e l d plot. At least one rain shower occurred during the 3-week duration of the t e s t .

k Three weeks after treatment. c

5

About 1 0 conidia/ml.



246


Granules are, however, an almost ideal vehicle for applying soilborne fungal weed pathogens. Weidemann (24, 25) used the alginate gel technique to formulate the endemic, soilborne fungus Fusarium solani f . sp. cucurbitae for the control of Texas gourd (Cucurbita texana) i n Arkansas soybean f i e l d s . This fungus causes a c o l l a r rot and root rot which leads to a rapid wilt and collapse of the weed. Alginate granules that were amended with the n u t r i t i o n a l adjuvant soyflour (2% w/v) and applied at a rate of 112 kg/ha and 224 kg/ha, and granules amended with ground oatmeal (2% w/v) and applied at 224 kg/ha gave >80% weed control within 6 weeks. Spores washed off the granules into the s o i l after r a i n f a l l , thereby contacting the target weed at the s o i l l e v e l and below to cause i n f e c t i o n . The number of fungal spores produced i s greatly Increased i n the s o i l when a n u t r i t i o n a l component i s incorporated in the formulation. Both pre- and postemergence (seedling stage) applications of the alginate granules were e f f e c t i v e . Soilborne Plant Disease Control Alginate granule formulations containing antagonist microorganisms have been an innovative and promising approach for the control of soilborne plant pathogenic fungi. The spherical, biodegradable granules can be applied to s o i l using conventional a g r i c u l t u r a l equipment• Fravel, et a l . (26) incorporated conidia and/or ascospores of i s o l a t e s of Talaromyces flavus, Gliocladium virens, Pen!ciIlium oxaIleum, or Trichoderma v i r i d e , or c e l l s of the bacteria Pseudomonas cepacia i n alginate granules with a pyrophyllite f i l l e r . The soilborne plant disease targets of these and similar biocontrol organisms are shown i n Table IV. Many crops worldwide are severely impacted by disease-causing microorganisms. Rhizoctonia s o l a n i , for example, i s a fungal pathogen of about 200 economically important crops. V i a b i l i t y of the biocontrol agents i n formulations gelled with calcium chloride and calcium gluconate were compared a f t e r 12 weeks and i t was found that calcium gluconate enhanced the survival of encapsulated bacteria and fungi better than the chloride. Bashan (19), however, found that calcium chloride was a s a t i s f a c t o r y gellant for use with bacteria when a secondary m u l t i p l i c a t i o n of the bacteria was induced by incubation of the gel beads i n fresh nutrient broth medium. Fravel, et a l . (27) have subsequently shown that an alginate/ pyrophyllite/T^. flavus formulation suppressed V e r t i c i l l i u m wilt Incidence i n potatoes under actual production conditions. A s i g n i f i c a n t amount of protection carried over to the following year without further treatment (28). Papavizas, et a l . (29) thoroughly studied alginate granule formulations of T^. flavus, p a r t i c u l a r l y the survival and multiplication of the fungus i n s o i l and survival of propagules during storage. Conidia i n alginate-bran granules gave the highest population density i n s o i l , but ascospores survived best i n storage (5-25'C).


19. CONNICK Table IV.

Living Biological Control Agents B i o l o g i c a l control agents Incorporated In alginate granules to combat soilborne plant pathogens

Biocontrol Agent


247

Plant Disease Target

Disease Pathogen

Bioassay Crop

Talaromyces flavus

Verticillium wilt

V e r t i c i l l i u m dahliae

potato, eggplant

Trichoderma hamatum

Rhizoctonia damping-off

Rhizoctonia solani

cotton, sugar beet

··

··

··

··

··

tt

II

Trichoderma harzianum Trichoderma v i r i d e Gliocladium virens Pseudomonas cepacia

·· Phythium damping-off

Phythium ultimum

soybean

Lewis and Papavizas (30) and Lewis, et a l . (31) f i r s t demonstrated that when an organic n u t r i t i v e f i l l e r , wheat bran, i s incorporated i n alginate granules with Trichoderma or Gliocladium instead of an inert clay, p r o l i f e r a t i o n of the biocontrol fungus i n s o i l i s s i g n i f i c a n t l y increased. These alginate granules containing a food base were capable of reducing the inoculum density of the pathogen Rhizoctonia solani and preventing damping-off disease of cotton and sugar beet seedlings i n different s o i l types* Shelf l i f e of the formulations was good. The Lewis process allows the use of wet fungal biomass of Trichoderma or Gliocladium d i r e c t l y from fermentation vessels. Interestingly, r e l a t i v e l y low amounts of wet biomass were as e f f e c t i v e as high rates when they were incorporated i n alginate granules (32). Fungal biomass i s an ideal inoculum for granules because i t can easily be prepared i n large quantities and because i t contains abundant chlamydospores, the hardy, r e s i s t a n t , and e f f e c t i v e propagule of these biocontrol fungi. The considerable body of encouraging r e s u l t s obtained with alginate granules containing biocontrol fungi applied to s o i l for the control of soilborne plant disease indicates a good commercial potential for these formulations i n selected applications. However, more research i s needed to further improve effectiveness and shelf life. Alginate Seed Treatments f o r Disease Control Placing a b i o l o g i c a l control agent d i r e c t l y on a crop seed insures i t s presence where the protection i s needed. F r a v e l , et a l . (28) incorporated two benefical bacteria together i n an alginatepyrophyllite seed c o a t L i n g » for soybean. , ggeu40fflpnas cepacia




248

suppressed postemergence damping-off disease and Rhizobium japonlcum e f f e c t i v e l y nodulated the soybean roots for nitrogen f i x a t i o n . A multi-purpose formulation such as this involving compatible microorganisms has obvious advantages, and i s worthy of additional research• Garber, et a l . (33) coated cotton seed with T. v i r i d e , G. virens, or Bacillus s u b t i l i s incorporated together with oat bran i n a hard, strong, adherent matrix of alginate-pyrophyllite. Positive seedling disease control was obtained. The biocontrol fungi were e a s i l y isolated from seedlings taken from plots where they were applied as seed coatings. Results obtained the following year (34) when the oat bran was s t e r i l i z e d before use were not as good. I t was postulated that a high, natural population of Bacillus spp. present i n the u n s t e r i l i z e d bran had provided an additional protection against Pythium spp. or R. s o l a n i . P a r t i c u l a r l y good results were obtained when fungicides were applied i n combination with the biocontrol seed treatments. Encapsulation of Nematodes Kaya and Nelsen (35) recently opened a new area of research i n insect control with the encapsulation of biocontrol nematodes i n alginate gel beads. In order to perform t h e i r function as biocontrol agents, the nematodes must somehow escape or be liberated from encapsulation to seek out hosts. Infective stages of the nematodes Steinernema f e l t i a e or Heterorhabditis h e l i o t h i d i s were encapsulated at a concentration of about 300 nematodes per gel bead (capsule) and fed to larvae of beet armyworm (Spodoptera exigua). The nematodes were released when the insect larvae b i t .into the g e l beads. The k i l l rate was excellent as long as adequate moisture was present to prevent desiccation of the nematodes (Table V). The f e l t i a e formulation maintained i t s population and i n f e c t i v i t y when stored f o r 8 months i n closed containers at 4 ° C Table V.

Control of beet armyworm (S. exigua) larvae exposed to nematodes encapsulated i n alginate gel beads a

Number of Larvae Dead

Nematode Genus

Alive

Heterorhabditis

2

35

Steinernema

0

38

36

0

Control

b

Larvae and gel beads were placed on moist f i l t e r paper. b

Gel beads without nematodes.

SOURCE: Data from ref. 35.



19. CONNICK


249

Infective stages of J5. f e l t i a e (-Neoaplectana carpocapsae) and H. h e l i o t h i d i s were also encapsulated i n alginate capsules by Poinar, et a l . (36). The f e l t i a e capsules were placed i n several habitats and, with adequate moisture, most of the nematodes were able to migrate out of the capsules by one week. When bacteria were abundant i n the surrounding environment, they decomposed the capsule matrix and accelerated release of the nematodes. Kaya, et a l . (37) recently devised a technique to place a quantity of f e l t i a e nematodes and a tomato seed into each alginate gel bead. Nematodes are released from the beads when the seeds germinate and are therefore already i n position to protect the seedlings from insect attack.. Successful bioassays with these gel bead formulations were conducted i n s t e r i l i z e d and nonsterilized s o i l against larvae of the greater waxmoth ( G a l l e r i a me Hone 11a). Insect larvae were k i l l e d even without seed present i n the soil-applied capsules which indicates that some nematodes managed to escape from the capsules through their own a b i l i t y . Calcium i s removed from complexation with alginate by the action of phosphate (19, 26) or c i t r a t e (37) buffer solutions which p r e f e r e n t i a l l y bind the calcium and s o l u b i l i z e the alginate matrix. In t h i s way, calcium alginate gel beads or granules can be dissolved so that entrapped organisms can be counted easily* It should be noted that phosphate f e r t i l i z e r would also act to weaken or degrade the structure of calcium alginate formulations i n s o i l and accelerate the release of active ingredients. Conclusions Alginate technology has proven to be useful f o r the formulation of a variety of b i o l o g i c a l control agents. Dried or hydrated calcium alginate matrices can protect l i v i n g organisms i n storage and i n the environment, yet permit the escape of entrapped agents to attack the target pests. Combinations of biocontrol agents or combinations of chemical and b i o l o g i c a l pesticides i n a single alginate formulation show great potential. With such combinations, a broader spectrum of pest control could be achieved from a single application. I t i s now time to study i n d e t a i l the economics of alginate formulations. Cost-effective formulations of biocontrol agents are of paramount importance for the success of biocontrol technology, and research i n this area needs to be increased. Literature Cited 1. 2. 3. 4. 5. 6. 7.

McNeely, W. H.; P e t t i t t , D. J. In I n d u s t r i a l Gums; Whistler, R. L., Ed.; Academic Press: New York, 1973, 2nd ed., pp. 49-81. Barrett, P. R. F. Pestic. S c i . 1978, 9, 425-433. Barrett, P. R. F.; Logan, P. Proc. EWRS 6th Int. Symp. Aquat. Weeds. 1982, pp. 193-199. Barrett, P. R. F.; Murphy, K. J. Proc. EWRS 6th Int. Symp. Aquat. Weeds. 1982, pp. 200-208. Kierstan, M.; Bucke, C. Biotechnol. Bioeng. 1977, 19, 387-397. Takata, I.; Tosa, T.; Chibata, I. J . Solid-Phase Biochem. 1977, 2, 225-236. Vorlop, K.-D.; K l e i n , J. In Enzyme Technology; Lafferty, R. M., Ed.; Springer-Verlag: New York, 1983; pp. 219-235.


250



8.

Livernoche, D., Jurasek, L.; Desrochers, M., Veliky, I . A. Biotechnol. L e t t . 1981, 701-706. 9. Scher, H. B. U. S. Patent 4 053 627, 1977. 10. Connick, W. J . , Jr. P r o c 6th I n t . Symp. Controlled Release Bioact. Mater. 1979, Sect. III, pp. 1-3. 11. Connick, W. J . , Jr. J . Appl. Polym. S c i . 1982, 27, 3341-3348. 12. Connick, W. J . , Jr. U. S. Patent 4 400 391, 1983. 13. Connick, W. J . , Jr. U. S. Patent 4 401 456, 1983. 14. Connick, W. J . , J r . ; Bradow, J . M.; Wells, W.; Steward, K.K.; Van, T. K. J . Agric Food Chem. 1984, 32, 1199-1205. 15. Bahadir, M.; P f i s t e r , G. Ecotoxicol. Environ. Saf. 1985, 10, 197-201. 16. P f i s t e r , G.; Bahadir, M.; Korte, F. J . Controlled Release 1986, 3, 229-233. 17. Bahadir, M. Chemosphere. 1987, 16, 615-621. 18. Jung, G.; Mugnier, J . Plant S o i l . 1982, 65, 219-231. 19. Bashan, Y. Appl. Environ. M i c r o b i o l . 1986, 51, 1089-1098. 20. Boyette, C. D.; Walker, H. L. Phytopathology 1985, 75, 183-185. 21. Walker, H. L.; Connick, W. J . , J r . ; Weed S c i . 1983, 31, 333338. 22. Walker, H. L.; P r o c South. Weed S c i . Soc. 1983, 36, p. 139. 23. Boyette, C. D.; Walker, H. L. Weed S c i . 1986, 34, 106-109. 24. Weidemann, G. J. Abstr. 24th Arkansas A g r i c Pest. Assoc. Meet. 1985, p. 24. 25. Weidemann, G. J. Abstr. 1986 WSSA Meet. 1986, 26, p.50. 26. Fravel, D. R., Marois, J . J.; Lumsden, R. D., Connick, W. J . , Jr. Phytopathology 1985, 75, 774-777. 27. Fravel, D. R.; Davis, J. R.; Sorensen, L. H. B i o l . Cult. Tests. 1986, 1, 17. 28. Fravel, D. R.; Lewis, J. A.; Lumsden, R. D.; Papavizas, G. C. Proc. 13th Int. Symp. Controlled Release Bioact. Mater. 1986, 13, 48-49. 29. Papavizas, G. C.; Fravel, D. R.; Lewis, J. A. Phytopathology 1987, 77, 131-136. 30. Lewis, J. A.; Papavizas, G. C. Plant Pathol. 1985, 34, 571577. 31. Lewis, J. A.; Papavizas, G. C., Connick, W. J . , Jr. D. S. Patent 4 668 512, 1987. 32. Lewis, J. A.; Papavizas, G. C. Plant Pathol. 1987, in press. 33. Garber, R. H.; DeVay, J. E., Wakeman, R. J. Proc. Beltwide Cotton Prod. Res. Conf. 1986, pp. 20-21. 34. DeVay, J. E.; Garber, R. H.; Wakeman, R. J. Proc. Beltwide Cotton Prod. Res. Conf. 1987, pp. 29-35. 35. Kaya, H. K.; Nelsen, C. E. Environ. Entomol. 1985, 14, 572574. 36. Poinar, G. O., J r . ; Thomas, G. M.; L i n , K. C.; Mookerjee, P. IRCS Med. S c i . 1985, 13, 754-755. 37. Kaya, H. K.; Mannion, C. M.; Burlando, T. M.; Nelsen, C. E. J. Nematol. 1987, 19, 287-291.

RECEIVED January 11, 1988


Formulation of Living Biological Control Agents with Alginate - ACS

Recommend Documents