microorganisms - microbiology and molecular biology...

SOIL PRODUCED ANTIBIOTICS-PLANT DISEASE AND INSECT CONTROL

J. H. STALLINGS1Agricultural Research Service, USDA, Beltsville, Maryland

The soil is the natural habitant of coutless diseases live in the soil, and it has long beenhordes of microorganisms, consisting of many known that the composition of the soil micro-kinds and types, living in a constantly changing flora can exert a controlling influence on them.environment. Most of them are active one way This effect is connected closely with microbialor another in reducing complex organic substances antagonism. When a soil has been sterilized,to simpler forms. Usually their existence pro- competition between microorganisms is reducedduces a healthy soil and a more favorable en- or eliminated; when such a soil is invaded latervironment for plant growth. The latter is brought by a parasite, the disease spreads much moreabout in part by increasing the food supply and quickly and is more virulent than in a naturalimproving the structure of the soil. Some of the nonsterilized soil. Thus, the microflora of the soilorganisms are injurious, however. They can cause acts as a barrier to the invader.a variety of diseases among cultivated plants. Organic substances high in carbonaceous mate-During recent years it has been discovered that rials appear to be particularly favorable to thethe organisms which produce the "wonder" development of antibiotic producing microorgan-drugs (e.g., penicillin) used in treating certain isms in the soil. The limited data available onhuman ills also produce these antibiotics in the this point suggest that it may have practicalsoil. Certain of these antibiotics have been found possibilities under field conditions in controllingcapable of destroying or inhibiting the develop- plant diseases and insects by using suitablement of a number of plant pathogens, viruses, cropping systems and the proper utilization of theand insect pests, both in the laboratory and under resultant plant residues.field conditions. It has been found that the most intensivePresumably these antibiotics are produced in microbiological activity occurs in the rhizosphere.

the soil for the protection of the microorganisms The microbiological population and activity alsoproducing them. The apparent purpose is to vary widely within different parts of the rhizo-enable the particular organisms to hold their own sphere. The antibiotic and associative inter-in competition with numerous others, perhaps by actions between groups of microorganisms areinsuring the production of an uninterrupted sup- thought to be intensified in the rhizosphere byply of food and energy. This is accomplished the effect of root growth and decay on the num-through the elimination or control of plant patho- bers and kinds present. This intensified activitygens, viruses, and insect pests that prey on their is believed to account for the production of sub-food producing plants. stances beneficial to higher plants when absorbedThese antibiotics may destroy the plant patho- by their roots. This intensified microbial activity

gens either directly by attacking them in the soil in the rhizosphere is thought to be related closelyor by acting systemically. In the latter case, the to other conditions existing in soils. There is aantibiotics, after being absorbed by the roots, vast difference in the microflora of the rhizo-are translocated to other parts of the plant, sphere of disease resistant and disease susceptiblerendering the penetrated tissues toxic (1, 2, 3). varieties and strains of crops grown in the sameCertain plant pathogens, viruses, and insect soil under identical conditions. Antibiotics pro-pests which invade or attack the plants are thus duced in the soil have prevented some plantdestroyed. diseases by inhibiting or destroying pathogenicMany of the organisms which cause plant microorganisms, either in the soil or systemically1 J. H. Stallings, formerly research specialist when absorbed by roots of higher plants.

with Soil Conservation Service, now with Agri- It appears that those genetic factors whichcultural Research Service, Soil and Water impart disease or insect resistance to a plant mayConservation Research Branch, U. S. Department do so by enabling that plant to produce a moreof Agriculture, Beltsville, Maryland. favorable microflora in the rhizosphere. The

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132 J. H. STALLINGS [VOL. 18

modified microflora of the rhizosphere of re- dition of Trichoderna lignorum to sterilized soilsistant varieties may be capable of producing simultaneously with the pathogen Rhizoctoniaantibiotics which destroy or inhibit the disease solani prevented the damping-off of citrus seed-producing germs, the invading viruses, or the lings (17). Numerous other instances of this typeattacking insect pests. of biological control of plant diseases have been

reported (18, 19, 20).ANTIBIOTIC PRODUCING SOIL MICROORGANISMS The effect of antibiotics operating in two orThe microflora of the soil is very complex. more combinations may be one of four types,

Almost all of the vast number of kinds and types namely, synergistic, additive, interfering, orof organisms are subject to numerous antagonistic indifferent, depending on the particular anti-as well as associative, or even symbiotic, inter- biotics involved (21, 22).relations. A radical change in the soil in one re- Meredith and Semeniuk (23) reported that 21spect is followed by equally radical changes in per cent of the strains of Actinomycetes isolatedmany other respects until new conditions of from two soils exhibited antagonism to Pythiumequilibrium are obtained. This is especially true of graminicola, which causes seedling root necrosis.the soil bacteria. If the type of food to support Under certain conditions the growth of P.one type of organism is exhausted, that which is graminicola was inhibited by the antibiotic pro-left will support some other kind. This shift in duced by streptomyces. Claviformin, gliotoxin,turn will cause numerous changes with far reach- penicillin, tyrothricin, eheiroline, and aspergillicing results (4). acid were effective in inhibiting the growth of

Tests by Lochhead and Landerkin (5) on the certain plant pathogenic bacteria and fungiinteraction of 90 strains of actinomycetes showed (24).that a wide range of cross-antagonisms existed Xylaria multiplex, an associated fungus, com-within a group of 11 strains found to be antago- pletely prevented the growth of Chalara quercina,nistic to Streptomyces scabies. These strains were the oak wilt fungus, when grown on laboratoryisolated by nonselective procedure from the media (25). Chloromycetin is synthesized byrhizosphere of potatoes grown in scab infected Streptomyces venezuelae and is active againstsoil which had been modified favorably by or- many bacteria (26). This organism produced anganic amendment. The possible importance of antibiotic in the soil which had been sterilizedthis in the study of specific antagonist-pathogen previous to its infestation with actinomycetes.relationships is indicated, as well as that of the Alfalfa hay turned under was effective in causing"rhizosphere effect" in modifying the microbial Streptomyces venezuelae to produce chloromycetinequilibrium in the soil. Some forms, such as the in the soil. Under selected conditions, Meredith'sActinomycetes spp., can produce such antibiotics actinomycete produced culture fluids toxic toas actinomycin, streptothrycin, and streptomycin many fungi (7). Musarin, the active principle,(6, 7, 8, 9, 10, 11, 12). These substances may be is a potent antifungal antibiotic. It is activebeneficial, neutral, antagonistic, or inhibitive to against several important parasites and may beother forms (13). They can destroy forms patho- useful in combating a number of fungus diseases.genic to cultivated plants, and they also may Certain antibiotic agents were found to exertproduce antibiotics with insecticidal qualities selective fungistatic and even fungicidal proper-(14). On the other hand, the same species of ties (27). Actinomycin had a strong effect uponmicroorganism can manufacture more than one Ceratostomella u1mi, the causative agent of theantibiotic. The different antibiotics produced by Dutch elm disease. Penicillin cured crown gallthe same species of microorganism differ greatly on Bryophyllum (28). Actidione was highly ac-from each other and affect a different range of tive against certain yeasts, including the fungalorganisms. pathogen Cryptococcus neoformans (29). Soils

It has been shown that antibiotic producing were found to contain substances of the actino-strains of Streptomyces rimou and S. aureomycin type which inhibit the growth of certainfaciens inhibited sensitive organisms in soil (15). bacteria in culture media (30). These substancesA streptomycin producing strain and a non- when isolated from the soil have both bacterio-producing mutant of S. griseus depressed the de- static and bacteriocidal effect upon certainvelopment of Bacillus subtilis (16). Penicillin had bacteria.similar effects on Pythium debaryanum. The ad- Ahmad et al. (6) isolated a new antibiotic,


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1954] SOIL PRODUCED ANTIBIOTICS 133

antimycin A, from cultures of a Streptomyces but also a bactericidal action upon certain bac-species that is toxic to many yeasts and other teria.fungi but is relatively inactive towards bacteria. Siminoff and Gottlieb (16) found that both aIts effect on several of the more sensitive or- streptomycin producing strain and a nonproduc-ganisms is easily observable at concentrations ing mutant of Streptomyces griseus depressed theof a few parts per billion. Waksman and Horning development of Bacillus sbtilis in unamended(31) isolated a number of fungi from soil which and tryptone amended soil cultures. In studyingwere antagonistic to bacteria. These fungi be- the stability of ten antibiotics in Bangshot sandlonged to nine distinct taxonomic groups. Some soils and in a neutral garden loam, Jeffreys (33)produced antibiotics rapidly on laboratory media. found that the rate of inactivation varied from

In studying the in vitro effect of aureomycin, soil to soil, but all exhibited a fair degree of sta-chloromycetin, streptomycin, dihydrostrepto- bility in some of the soils. Gottlieb and Siminoffmycin, penicillin, and bacitracin on bacterial (26) found that chloromycetin could be producedstrains of several species, Spencer (22) found that by Streptomyces venezuelae when grown in un-each antibiotic produced destructive action on amended soil. Its production was greatly in-the majority of the organisms in a sensitive creased by the addition of tryptone and onlystrain. Dubos and Hotchkiss (9) reported that slightly by the addition of green alfalfa hay. Oatseveral species of aerobic sporulating bacilli straw and starch were ineffective. Gottlieb et al.isolated from soil, sewage, manure and cheese- (34) reported that both actidione and clavacinas well as authentic strains obtained from type inhibited the growth of sensitive microorganismsculture collections-exhibited antagonistic ac- in the soil. Actidione was produced in steriletivity against unrelated microorganisms. Cul- soil by Streptomyces griseus when soybean mealtures of these organisms yielded tyrothricin was added. The amount of actidione synthesizedwhich was bactericidal for most gram positive in such soil depended on the concentration ofand gram negative microbial species. soybean meal.

AspergiUus clavatus was antagonistic to BacillusANTIBIOTIC PRODUCTION IN SOIL subtilis when sterile soil was infested with both

Trichothecin was synthesized by the fungus of these organisms (34). Clavacin could not beTrichothecium roseum when grown in a variety found in soils infested with A. clavatus when dif-of unsterilized soils (32); clay soils had a higher ferent carbonaceous materials were added totrichothecin producing capacity than sandy the substrate. The presence of clavacin in soilsoils. It was adsorbed strongly by soil, but very caused the rise of a resistant microflora in thelittle biological breakdown occurred. It has been soil.demonstrated that chloromycetin can be pro-duced in sterilized soil by Streptomyces vnezuelae(26), and that the production and activity of In preliminary experiments with severalthis antibiotic were greatly increased by the plant species, Brian et al. (2) immersed the cutaddition of tryptone. ends of shoots in an aqueous solution of griseo-Johnson (20), after studying four types of fulvin (10 or 100 jsg/ml). They reported that

bacteria antibiotic to certain smuts and fungi, griseofulvin could be detected in the upper leavessuggested that antibiotic processes occur in in 7 to 14 days. These investigators also foundnature. Martin and Gottlieb (15) found that that griseofulvin acted systemically, practicallyantibiotic strains of Streptomyces rimosus and to control the fungus parasite, Botrytis cinerea,S. aureofaciens inhibited sensitive organisms in when grown in water culture nutrient containingsoil; the amount of antibiotic necessary for the griseofulvin. It also controlled Alternaria solaniinhibition of a sensitive organim in soil was when used in nutrient solution on tomatoesdependent upon the pH of the soil solution. By grown in sand culture. Griseofulvin showed un-the use of ether as an extracting agent, Waksman doubted activity as a systemic fungicide. Theseand Woodruff (12) demonstrated that soils investigations also produced results which indi-contained substances of the actinomycin type, cated griseofulvin was produced in both ster-which inhibited the growth of certain bacteria ilized and unsterilized soil.in culture media. When isolated from the soil, Mitchell et al. (3) found that the applicationthese substances had not only a bacteriostatic of a small amount of streptomycin sulfate to


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stems of primary leaves of bean plants, prior soil heavily with a species of Trichoderma. Gardento inoculating the leaves with the halo blight peas showed a similar but less marked response.organisms Pseudomonas midicaginis var. phaseo- These workers thought the Trichoderma maylwola, prevented them from developing symptoms have had an antagonistic effect on the growthof the disease. Similar plants inoculated in a like of Rhizotonia and Pythium. The presence of anmanner developed very mild symptoms of the active principle, toxic or lethal to Rhizoconia,disease when dihydrostreptomycin sulfate was was demonstrated in a liquid medium in whichapplied to their stems prior to inoculation. These Trichoderma had grown for five days. The filtrateantibiotics apparently were absorbed by the from a five day old Trichoderma culture was lethalstems and translocated upward into the primary to Rhizctonia when used both in full strength orleaves in sufficient amounts to prevent growth when diluted to 40 per cent or less with nontoxicand development of the organs. Similar treat- diluents. Rhizoctdnia failed to grow in a freshlyment with these antibiotics prevented the de- prepared filter sterilized filtrate from a fivevelopment of the common bean blight caused day old Trichoderma culture but grew luxuriantlyby Xanthomonas phaseoli (E.F.Sm) Dowson. in a similar filtrate after the toxic principle hadStreptomycin sulfate was absorbed by the stems been inactivated or destroyed.of bean seedlings and translocated to trifoliate Patulin, previously obtained from Penicilliumleaves 6 to 8 inches from the point of treatment patulum Bainier, was shown by Anslow et al.in sufficient amounts to reduce the incidence of (39) to be a metabolic product of the apple-rotthe infection and severity of symptoms produced organism, P. expansum (Link) Thom. It com-by the blight organism. pletely inhibited the growth of various species

Rhide et al. (35) found that of the 20 species of Pythium at dilutions of approximatelyof Xanthomonas tested, X. cassiae and X. begoniae 1:400,000.were not inhibited by any of the actinomycetes. Anwar (19) found that 48 out of a total ofThe other species were inhibited by one or more 86 isolates of bacteria, actinomycetes, and fungicultures of the actinomycetes. Of the 64 cultures from soil were antibiotic to Helmirnthosporiumof actinomycetes under study, seven proved sativum and 12 to Fuwarium lini when grown onantibiotic to three or more species of the bacteria potato-glucose agar. The metabolic products oftested. some of the bacteria, actinomycetes, and fungi

Zoril (36) protected carnations against attacks inhibited the growth of H. sativum when addedfrom Fusarium culmorum and F. oxysporum f. to agar medium.dianthi by applying Trichoderma ligorum to the A soil isolate of Bacilus subtilis, when addedsoil in which carnation plants were growing one to steamed soil with H. sativum, completely pro-week prior to the addition of the pathogens. The tected barley seedlings against root rot. B.organism was parasitic on the fusaria. eubtilis protected field plots of barley from heavyIn small scale greenhouse and field experiments infection by Helminthosporium. Trichoderma

with Helminthosporium victoriae blight of Vicland lignorum and Penicillium sp. also gave partialoats, Leben et al. (37) showed that a partially protection from disease. Whiffen (40) reportedpurified preparation of the antibiotic helixin B that actidione, produced by Streptomyces grisew,resulted in disease control in most tests. In similar was highly active against certain yeasts, includ-experiments with Wisconsin Barbless barley, ing the fungal pathogen Cryptococcus neoformans.seed treatments with the antibiotic controlled Young and Brandt (25) found that Xylariathe seedling blight incited by H. 8atWvum. Seed multiplex completely prevented the growth oftreatment with the antibiotic was effective in Chalara quercina (oak wilt disease) in agar plates.limited field tests for controlling wheat bunt, oat Sugar beet pulp incorporated into a soil con-smut, and covered smut of barley. Helixin B taining Phytophthora parasitica and inoculatedpreparations acted across an air space to inhibit with Peniciium patulum prevented damping-offthe germination of spores of a test fungus. of tomato seedlings (41). In this study, antibioticsZaumeyer (38) successfully stopped and con- were produced in a partially sterilized soil, and

trolled the halo blight of beans by spraying the Aspergillus terreus produced them in sterilizedfoliage with water containing small amounts of soil. Timothy hay was a satisfactory source ofstreptomycin. Allen and Haenseler (18) reduced organic matter for antibiotic production in auto-seed decay and damping-off by inoculating the claved soil.


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19541 SOIL PRODUCED ANTIBIOTICS 135

Brown and Boyle (28, 42) cured crown gall on ing root rot of Pinus sylvestris and P. nigra var.Bryophyllum with crude penicillin. The galls cLabia, was suppressed on laboratory mediumwere punctured in a number of places, and peni- by Trichoderna viride.cillin soaked antiseptic cotton was wrapped Cooper and Chilton (46) reported that thearound them. The cotton was wetted frequently four major sugarcane soils of Louisiana showedwith crude penicillin. These workers found in a consistent difference in the number of Actino-laboratory studies that penicillin depressed mycee spp. antibiotic to Pythium arrhenomas,Erwinia carnegieana Stranding, which has one of the important fungi causing root rot ofcaused extensive destruction of the giant cactus sugarcane. The number of these organisms pres-in Arizona and New Mexico. Evidence was ent was in direct relation to the amount of or-obtained indicating that Corynebacterium spede- ganic matter in the soil; the Yahola silt loamsonicum was also susceptible to the action of had the highest number, the Yazoo silt loamspenicillin. Good control of damping-off of citrus the next, the Iberia silt loams and first terraceseedlings due to Rhiwoctonia solani was secured soils were third, and Sharkey clays the lowest.by adding Trichoderma sp. to a sufficiently acid, Comparative yield tests during five years withsterilized soil (17). Trichoderma koningi and T. sugarcane on the various soils gave yields cor-album, as well as all isolates of T. lignorum relating with the number of species of antibiotictested, were found to attack the Rhieoctonia. Actinomyces. In greenhouse tests in sterile soil,A number of fungi and bacteria exerted a certain of the Actinomyces increased in the soil

marked antagonistic action against the pathogen and reduced root rot in corn.Ophiobolus graminis on infected wheat seedlings Stessel et al. (47) isolated 170 antagonistic(27). In addition to the living cultures, the cul- organisms from approximately 70,000 coloniesture filtrates were also effective. Growth of obtained from soil during a screening test de-Helminthosporium sativum and Fusarium gra- signed to discover antagonists suitable for plantminearum on sterilized soil was suppressed com- disease control. Approximately 80 per cent ofpletely by adding small amounts of unsterilized these were actinomycetes. Of the one hundredsoil or by simultaneous inoculation with harmless and two kept for further studies, a few organismsfungi and bacteria. Wheat seedlings produced were antagonistic to four or more of the 10from seeds inoculated with this soil were free phytopathogens tested.of infection. Although H. sativum sporulated Gregory et al. (48) obtained from 26 samplesrapidly in sterilized soil, it did not do so in of soil, peat, and chaff, 31 bacteria, 29 actino-unsterilized soil; sporulating was inhibited in mycetes, and 14 fungi which were stronglythe unsterilized soil by microorganisms. antagonistic to Pythium debaryanum. Of theLeben and Keitt (43) demonstrated antibiotic most potent cutlures, three actinomycetes and

properties of antimycin against certain phyto- one bacterium were innocuous to Rhizobiumpathogens and found that it was an extremely meliloti and R. trifolii. The antibiotics, actidionepotent fungicide, producing inhibitory effects and fradicin, were also active against Pythiumagainst Nigrospora aphaerica. A species of Strepto- sp. but inactive against Rhizobium sp. Clavacinmyces was antagonistic on agar to the 33 fungi (Penicillium patulum) and gliotoxin (Trichodernatested but was not antagonistic to a number of lignorum) were toxic to species of both Pythiumbacteria. The antibiotic, which was tentatively and Rhizbium.named antimycin, was produced in shake flasks Penicillium patulum, Trichoderma lignorum,and by tank fermentation. Hessayon (44) found Streptomyces sp., strain A67, and Bacillus sp.,that small concentrations (0.00018-0.0018 strain B6, were the antibiotic producers. Eachunits/ml) of trichothecin smulated the growth organism produced potent antibiotic activity inof Fusarium oxysporum var. cubense when grown sterile soil containing suitable organic material.on laboratory media. In higher concentrations Bacillus sp. B6 produced more activity in suit-(0.0018-10 units/ml) the effect was fungistatic. ably treated soil than in liquid culture. P.Above these amounts there was a killing effect, patulum and Bacillus sp. B6 produced appreci-and with 44 units/ml the effect was wholly able amounts of antibiotic in nonsterile soil con-fungicidal. He was able to produce trichothecin taming 0.5 per cent soybean meal, 0.5 per centin a sterile soil by Trihotheium roseum. Risbeth glucose, and 0.2 per cent corn steep liquor. Whif-(45) found that Fomes annosu, the fungus caus- fen et al. (49) found that the growth of the fungal


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pathogen, Cryptococcus neoformans, was inhibited fertilizer are credited with this change (55). Itin a 1:100 dilution of a beer of Streptomyces is contended that the fertilizer stimulates fastergrseus containing 130,ug per ml of streptomycin, growth of the seedlings. Incorporation of cropthat the organism was not inhibited by 285 jsg residues and trashy fallow act mainly throughper ml of highly purified streptomycin. the complex microbiological activities that pre-

In testing a group of 197 cultures of actino- vent a flare-up of the parasitic soil fungi (Pythiummyces for their antibiotic activity against sp.) at the time the host plants are in the seed-Ceratostomella ulmi, Lechevalier et al. (50) found ling stage.a strain of Streptomyces griseus which produced The antibiotic griseofulvin is produced by thean antibiotic candicidin that was active against fungus Penicillium nigricans, which is abundantyeasts, yeast fungi, and C. ulmi. Crude candicidin and widely distributed in natural and agriculturalwas fungicidal against growing and resting cells soils (56, 57), as well as by the less commonof Candida albicans in the concentration of 1 to Penicillium griseofulvum (2). It can be taken5 mcg/ml and had no injurious effects upon the into plants through their root systems and thengermination of pea seeds in concentration of can be translocated to other parts. It has marked125 mcg/ml. Spraying young bean plants once a fungistatic properties and is active as a systemicweek with an aqueous suspension of crude candi- fungicide. Similar activity is possessed by certaincidin (660 mcg/ml) resulted in a decrease of aryloxyaliphatic acids (58) and 4-nitrosopyrazolesmildew infection. (59). The use of systemic compounds was effec-

Sanford and Bradfoot (51) working with 26 tive in treating Dutch elm disease (60,61).cultures of fungi and 40 of bacteria, nearly all ofwhich were isolated from the soil, found six CARBOHYDRATE LEVEL AND DISEASE CONTROLcultures of fungi and 15 of bacteria that sup- Soybeans grown as a cover crop and incor-pressed the pathogenicity of Ophiobolus graminis porated into strawberry root rot soil caused ato a degree varying from almost zero to a 10 striking reduction in the incidence of root rot andper cent infection rate. It was concluded that a drastic shift in the bacterial equilibrium of thethe toxicity of the living cultures or of their soil according to West and Hildebrand (29, 62).filtrates was the chief factor in suppressing the A red clover cover crop turned under had littlevirulence of the pathogen, and that many soil effect on either the severity of the disease or theinhabiting fungi and bacteria are effective in sup- general microflora of the soil. Their study ofpressing the pathogenicity of Ophiobolus gra- "rhizosphere effects" revealed that the charac-minis. By the addition of other fungi to the soil, teristic differences between the resultant bac-Luijk (52) found that it was possible to inhibit terial equilibrium of the soils in which the twothe parasitism of Pythium volutum on Agrostis leguminous plants were grown could not bestolonifera. Pullularia pullulans formed sub- attributable to influences exerted by the latter instances growth inhibiting to Pythium species the living state. However, the bacterial typeswhen grown on laboratory media. The rotting favored during decomposition in experimentalof grass seedlings by Pythium was inhibited by cultures of tissues of red clover and soybean,products formed by Pullularia, and neither each inoculated with root rot soil, were identicalseeds nor the seedlings were damaged by these with those isolated from root rot soil with whichsubstances. Pythium species were antagonistic red clover and soybean, respectively, had beenmutually in nonsterile conditions. incorporated. In contrast with the putrefactive

Applications of fresh organic matter destroyed decomposition of red clover, soybeans apparentlycertain plant pathogens originally in the soil underwent a carbohydrate breakdown that could(53). Corn, normally immune to Phymatotrichum be reproduced essentially in culture by the sub-root rot, was attacked and killed readily when stitution of glucose for soybean tissues. Beneficialgrown in sterile culture without its normal com- changes in the bacteriology of actual root rotplement of root surface bacteria (54). Browning soils could be induced by the decomposition ofroot rot of cereals, formerly a serious disease of pure carbohydrate in the place of soybean. Thesummer-fallow crops on the Canadian prairies, favorable alteration in the bacterial equilibriumis seldom encountered. A change to farming was accompanied by a corresponding modificationpractices of returning practically all of the crop of the fungus flora such that potentially patho-residues to the land and liberal use of phosphate genic forms were replaced by presumably in-


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nocuous ones. These carbohydrate treated soils with roots. Most of the soil isolates did not uti-were capable of producing strawberry plants with lize sucrose or hydrolyze starch, whereas thewell developed healthy root systems. The ability majority of root isolates fermented the moreof soybeans to control strawberry root rot, complex sugars.therefore, seemed to depend primarily on acarbohydrate type of breakdown in diseased soil ANTIBIOTICS AND VIRUS CONTROLcausing a highly favorable shift in the microbial Antibiotics produced by a number of soilequilibrium. The decomposition of red clover, on inhabiting organisms have been found to inhibitthe other hand, did not induce these salutary certain plant viruses. Two general theories,effects under the same conditions. accounting for the mechanism by which this is

After altering the amount and source of carbon accomplished, have been advanced. Accordingin the laboratory media, Luijk (52) concluded to the first, the inhibitor enters into a loose butthat carbon was the most important substance reversible combination with the virus (65).causing inhibitory effects. He thought that the According to the second, the inhibitor alters theinhibiting substance was a metabolic product of physiology of host cells so they no longer supportthe carbon compounds. virus multiplication (8, 11).

Grossbard (63) showed that sugar beet pulp Bawden and Pirie (65) found that the addi-was a very satisfactory material for the production of trypsin immediately reduced the infec-tion of an antibiotic by Penicillium patulum. tivity of potato virus "X" without affecting theP. patulum produced an antibiotic when cultured flocculating power with antiserum. Incubationon sterilized soil to which either wheat straw or with crystalline preparations of trypsin and pep-glucose or sugar beet pulp was added. Aspergillus sin destroyed both the infectivity and the powerterreus and Streptomyces antibiotic also pro- of reacting with antiserum.duced antibiotics when grown on wheat straw. Filtrates of 5 different strains of NeurosporaThe susceptibility of cotton plants of fruiting sitophila and one strain of N. crassa were testedage to Phymatotrichum root rot, both in potted for ability to inhibit infection by Southern beansoil and in the field, was related to the carbo- mosaic, tobacco mosaic, and tobacco necrosishydrate content of the bark (54). With increasing viruses by Slagle et al. (11). All filtrates werecarbohydrate concentration, resistance was in- highly effective in reducing the number of infec-creased, and plants sufficiently high in carbo- tions, depending upon the host plant used. Ithydrates withstood attack. was effective immediately after mixing with virus,The correlation between carbohydrate con- no time reaction being involved. It inhibited

centration and the resistance of cotton to root infection whether applied directly to the virus orrot was believed to reflect antibiotic protection to host-plant leaves either before or up to 30at the higher carbohydrate level. The micro- minutes after inoculation, and when appliedbiological equilibria on the surface of cotton roots to the leaf surface opposite to that on which thewere altered markedly as the carbohydrate con- virus was inoculated as well as to the same sur-centration within the roots was increased. The face.number of certain organisms tended to increase Trichoderma sp. grown in liquid medium pro-through successive carbohydrate levels whereas duced a substance which had caused up to 90others decreased. The existence of an important per cent reduction in the infection capacity ofinteraction was demonstrated between root- the tobacco mosaic virus, measured in number ofsurface saprophytes and the parasitic activity local lesions on Nicotiana glutinosa half leafof the root fungus, Phymatotrichum omnivorum, inoculation (10). Under the experiments, theby means of experiments with maize. After fungus was grown in liquid medium and theinoculation with P. omnworum, maize plants filtrates used in inactivation tests on the virus.growing on sterile sand-bentonite substances It was concluded from the fact that the liquidwere rapidly attacked and killed. The roots of filtrate showed inactivation power after only twomaize plants on otherwise similar but nonsterile days of culture, that the inactivation is of thesubstrates remained healthy even though paral- nature of a secretion of the fungus into the liquidleled for long distances by strands of the fungus. medium. The inactivation proceeded within two

Clark (64) noted that the Pseudomonas types minutes after mixture of the culture extract withof bacteria from soil differed from those associated the virus; no better results were obtained if the


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mixtures were allowed to stand for a longer period substances are assimilated by higher plantsof time. through their roots and are systemic in nature.

Stanley (66) prepared a trypsin-virus solution Kido and Spyhalski (14) isolated antimycin Awhich when sprayed or rubbed on the leaves failed from cultures of an unidentified species of Strepto-to produce lesions on plants of Phaseolus vulgaris myces. The initial tests showed that the antibioticbut produced many lesions when tested on plants caused mortality to some insects which ingestedof Nicotiana glutinosa having about an equal the material. The toxicity of antimycin A wassusceptibility to untreated virus. He concluded not confined to members of the Insecta, as itthat this showed that trypsin in some way affects showed efficacy for the control of the red spiderthe cells of plants of P. vulgaris. Trypsin spread mite, Tetranychys sp.or rubbed on the leaves of plants of Nicotiana Davich and Apple (74) obtained satisfactoryglutinosa, N. tabacum var. Turkish, and Phaseolus control of aphides on peas by use of systemicvnlgaris markedly lowered their susceptibility insecticides. The systemic insecticides used wereto virus. A number of other viruses were affected octamethyl pyrophosphoride, trialkyl seleno-similarly by trypsin. They were a masked and a phosphate and trialkyl thio-phosphate. Theyyellow strain of tobacco mosaic, aucuba mosaic, were effective in controlling pea aphides whensevere etch, tobacco ring spot, ordinary cucum- used in proper amounts and applied to the soilber mosaic, and a yellow strain of cucumber as a spray prior to planting the peas or to themosaic virus. Trypsin was thought to inhibit leaves of the growing plants. Beck (75) reportedinfection by killing injured cells that otherwise that antimycin A had insecticidal possibilitiescould serve as entry points for virus (67) or by for some species of insects and mites. The anti-forming a loose complex with the tobacco mosaic biotic inhibited either the succinoxidase systemvirus (65). It reduced the infection of tobacco or some other essential step in the oxidativemosaic virus, and its effect was immediate. metabolic cycle of cockroaches. Inhibition of the

oxidative cycle readily explains the depressionANTIBIOTICS AND INSECT CONTROL of oxygen consumption by the poisoned insects.

Recent discoveries, relating to the production SYNERGISMin the soil of antibiotics by microorganismsand of the systemic action of certain insecticides Bigger (21) reported that the antibacterialwhen absorbed by plants (1, 68, 69, 70, 71, 72), action of six substances-sulfathiazole, penicillin,may lead to other discoveries showing nature streptomycin, chloromycetin, boric acid, andhas built into the soil, by means of organic matter p-aminosalicylic acid-acting in pairs on Escher-and microorganisms, a system which may be ichia coli in sympathetic medium was synergisticutilized to protect plants against diseases and in most cases. Synergism was demonstrated withinsects. It was shown earlier in this paper how every combination except one; antagonism wassome of the antibiotics that are produced in the demonstrated with five combinations. In onlysoil function in keeping certain soil plant patho- three cases was it marked and permanent, andgens from running rampant. The possible role in only one of these (boric acid and sulfathiazole)of these antibiotics in controlling insects is no was each substance antagonistic to the other.less intriguing. A study in vitro effect of penicillin and baci-

Insecticidal qualities of certain plants have tracin mixtures against several strains of a- andbeen known for centuries. To date, more than y-hemolytic streptococci indicated synergistic2,000 species of higher plants are said to have action between the two antibiotics instead ofvalue as insect killers (73). The active principle merely an additive effect (76). The synergisticincludes alkaloids, pyrethrins, wilfordine, nico- effect varied from strain to strain. It was verytine, anabasine, sesan, oils, asarinin, nornico- pronounced with some cultures while less dra-tine, glycoside, conine, scarbin, and others. The matic with others.insecticidal principle may be present in the leavesand leaflets, roots, bark and wood. The insects RH1ZOSPHERE AND MICROBIAL ACTIVITYaffected by these insecticides include a wide The rhizosphere is shedding light on the im-variety. Only recently, however, was it discovered portance of microorganisms in the production ofthat certain microorganisms in the soil produced crops as its nature and function become moreantibiotics of insecticidal qualities and that clearly understood. It has been shown that plant


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roots support on their surfaces a population of fect its capacity to thrive and exert its antibioticbacteria which differs from that of the surround- action. To be effective, an antagonistic microor-ing soil. It is of much greater density and differs ganism must be able to maintain its existencewidely in the type of its dominant species. Roots when exposed to the competition and antagonismhave been shown to provide the food supply of of the indigenous soil microflora, as well as tothe rhizosphere organis, partially in the form exert its antibiotic action when the microbialof soluble compounds which are excreted and balance is shifted and different competitions andpartially as cellular structure such as root caps, antagonisms come into play through action ofroot hairs, rootlets and epidermal and cortical the growing plant.cells, all of which are being sloughed off. The Lohhead and Thexton (79) found that onerhizosphere microflora has been shown to vary of the most characteristic rhizosphere effectswith the stage of growth of the plant and with on bacteria of different nutritional requirementsthe species and even with the variety of the was the preferential stimulation of bacteriaplant; indications are that it is probably of requiring amino acids for mum growth.importance in several respects (53). Organisms for which amino acids were eitherThe rhizosphere is a unique zone, exerting on esetia or stimulative were increased propor-

many soil orgisms a powerful stimulation tionately in the rhizosphere. No similar effectwhich varies with the type, variety, age and was noted with reference to bacteria- respondingvigor of the plant and the type, treatment and to growth factors.moisture content of the soil in which it grows Three main processes are believed to take(77). Any factor contributing to a modification place in the complex and intricate relationshipsof growth and physiological behavior of the plant between the microorganisms of the rhizospherethen may be expected to initiate changes in the and plant root (2). Excretion of soluble nutrientsroot microflora. Tnasmuch as the nutrient material and sloughing off of dead tissues by the rootscoming from the roots is primarily responsible for encourages the development of a microflorathis immediate microbial concentration, this greater in numbers and qualitatively differentmantle of microorganisms attached to the root from that of soil farther away from the roots.surface is perhaps the most important part of This higher level of microbial activity is thoughtthe rhizosphere. to intensify the antagonistic and associative

Bakerspigel and Miller (78) found bacteria to interactions between groups of microorganismsbe more abundant in the rhizosphere of iris, in the rhizosphere. Also, it is thought to influencebeans, and potatoes than in soil more distant the development of the plant through its effectsfrom the rhizosphere. The rhizosphere microor- on the roots.ganisms seemed about as sensitive to antibiotics In a study carried on withdised and healthyas from control samples. Tests by Lochhead and soils, Timonin (80) found that a susceptibleLanderkin (5) on the interaction of 90 strains of variety of oatsharbored in its rhizosphere a denseractinomycetes, isolated by nonselective pro- population of manganese oxidizing, casein hydro-cedure from the rhizosphere of potatoes grown lyzing, and denitrifying bacteria than the rhizo-in scab infested soil which had been modified sphere of a resistant variety when grown in thefavorably by organic amendment, showed that same soil under identical conditions. Furthermore,a wide range of cross-antagonisms existed within it was found that, on application of soil fumigants,a group of 11 strains, found to be antagonistic such as chloropicrin, cyanogas, calcium cyanide,to Streptomycee 8cabies. The possible importance and formaldehyde, the bacteria capable ofof this in the study of specific antagonist-patho- oxidizing manganese were greatly reduced orgen relationships is indicated, as well as that of completely eradicated. The plants grown in suchthe rhizosphere effect in modifying the microbial soils were free from symptoms of manganeseequilibrium in soil. deficiency disease and showed a marked increaseThe rhizosphere microflora varies both quan- in yield of grain. Application of straw mulch

titatively and qualitatively from that of soil resulted in a denser population of manganesefarther away from the plant as a rhizosphere is oxidizing and cellulose decomposing organisms,created following germination of a seed. A given and more severe symptoms of the disease thanantagonist, present in or added to the soil, finds the untreated soil.itself in a changed environment. This might af- In comparative studies of the rhizosphere of


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resistant and susceptible varieties of flax, Timonin exist between different varieties of flax and(81) showed that the incidence of species of tobacco.Alternaria, Cephalosporium, Fusarium, Helmin- Thom and Humfeld (86) reported considerablethosporium, and Verticillium was relatively variations in absolute counts and the rhizosphere:lowered and that of species of Mucor, Clado- soil (R:S) ratios of rye in three different soils.sporium, Penicillium, and Trichoderma increased A Keyport clay loam gave ratios of 6 and 5 forby the "rhizosphere effect" of the resistant variety fungi and bacteria as compared with 15 and 16as compared with the susceptible variety or on roots of plants in a Collington fine sandy loam.control soil. He also found that when the solu- In a study of corn growing in different soilstions in which plants had been grown were allowed varying from pH 4.5 to 8.1, bacteria were foundto diffuse through collodion membranes into the to be more numerous under slightly acid orsoil, the microbial activities in the vicinity of neutral conditions, and fungi were more abundantthe membrane were stimulated in a manner on roots in the most acid and most alkaline soil,analogous to that in the natural rhizosphere of whereas actinomycetes were not affected bycorresponding plants. The solution after growth soil differences. Adati (87) showed that, on theof the susceptible variety, when added to liquid whole, R:S ratios were highest on loam withor solid media, induced a greater stimulation of sand, clay, and humus soils next in order ofgrowth of Fusarium and Helminrthosporium than decreasing effectiveness. This order held for thethe solution after growth of the resistant variety. three major groups of organisms studied. TheChemical analysis indicated that the solutions influence of soil type on the rhizosphere micro-after growth of resistant variety contained from flora was observed also by other investigators25 to 37 mg hydrocyanic acid per plant grown, (78).whereas the solution after growth of the suscep- Chromogenic forms occurred in greatest rela-tible variety contained only a trace. Potassium tive abundance in tobacco rhizospheres; othercyanide when added to Crone's solution exerted plants showed similar though not such strikingan effect on the growth of the same fungi analo- differences (73). A somewhat more active micro-gous to that produced by the solution after growth flora was observed in the rhizosphere of suscep-of the resistant variety. tible varieties of flax and tobacco. Bacteria re-

Differences in rhizosphere effects may be quiring or stimulated by amino acids were rela-obtained also with different varieties of one tively more abundant in the rhizosphere of bothplant species. Obraztzova (82) found that the flax and tobacco plants than in the control soil,variety of tea-bush did not influence numbers of especially in the rhizospheres of susceptible varie-microbes but did affect their quantitative corm- ties (84). Similar observations were made withposition. Timonin (81) noted higher numbers of bacteria requiring growth factors in addition.bacteria and, to a lesser extent, of fungi in the Working with flax, Timonin (88) reported thatrhizosphere of varieties of flax and tobacco plants of 19 genera isolated, nine were obtained fromsusceptible to soil-borne plant pathogens, though both rhizosphere and control soil and ten fromfree from the disease, than in those of correspond- the rhizosphere only. Certain genera-Alter-ing resistant varieties. Data by Lochhead and naria, Aspergillus, Cephalosporium, Fusarium,co-workers (83, 84, 85) support this observation. Helminthosporium, and Verticilliumr-were nu-It was suggested that these results reflected in- merically more abundant in the rhizosphere of aherent differences in physiological function, mak- variety of flax susceptible to root rot, whereasing, in the case of susceptible plants, conditions others, such as Mucor, Cladosporium, Hymenula,somewhat more favorable for general bacterial Penicillium, Scolecobasidium, and Trichoderma,development. In this connection the interesting were more abundant in the rhizosphere of awork of Eaton and Rigler (54) showed that as resistant variety.the carbohydrate content of cotton roots in- In the rhizosphere, the phenomena of associa-creased the total bacterial count on the root tion, antagonism, and competition for oxygensurface decreased, suggesting that it is possible and food are perhaps even more intense than into produce within a single variety of cotton dif- the soil proper, owing to the denser populationferences in bacterial numbers and root rot sus- at the root surface and its greater physiologicalceptibility somewhat analogous to those that activity. Here the influence of root physiology


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and excretions may play an important role, of -46 and +10 for rhizosphere soil apart fromdirectly by stimulating or repelling the pathogens the root, respectively, as compared with +44or indirectly by affecting the rhizosphere micro- and +35 for steam treated soil. General agree-flora (89). Lochhead and co-workers (79, 85) ment was noted between this index and severityshowed that plants susceptible to certain root of the disease. No such correlation, however,rots induce a greater rhizosphere effect than cor- was observed in relation to manganese defi-responding resistant varieties, suggesting that ciency disease of oats (80). Hildebrand and Westresistance to a certain disease may be linked suggested that the organisms responsible forwith a selective action of root excretions upon lowering the bacterial balance index may actuallythe saprophytic soil microflora. This favors types be involved in production of the disease symp-whichmay be more and in other cases less antago- toms. However, Katznelson (84) pointed outnistic towards pathogenic organisms (85). In that it was possible that these organisms werethis connection, it was demonstrated (88) that a secondary rather than primary invaders, increas-wilt resistant variety of flax excreted hydro- ing in abundance on the root following infectioncyanic acid into the surrounding medium, thus by other organisms, such as nematodes or fungi,exercising a selective power upon the fungus or as a result of treatment and thus giving a lowflora of the rhizosphere. The cyanide depressed bacterial balance index.pathogenic fungi, such as Fusarium and Hel- West and Hildebrand (29) and West and Loch-minthorporium, but appeared to favor Tricho- head (92) showed, in addition, that the decom-derma viride, an organism mentioned frequently position of soybean or glucose in root rot soilfor its ability to suppress development of other or the addition of acetic acid resulted again in afungi (27,90,91). On the other hand, by-products favorable alteration of the bacterial equilibriumof growth of a susceptible variety stimulated and permitted growth of strawberry plants withgrowth of two pathogens. Eaton and Rigler well developed healthy root systems. These(54) showed that cotton plants with low carbo- treatments also modified the fungus flora on thehydrate concentration were attacked most roots of strawberry seedlings grown in the treatedseverely by Phymatotrichum omnivorum, had soil so that potentially pathogenic forms (Fusar-largest number of bacteria and lowest number of ium, Rhizoctonia, Cylindrocarpon) were replacedblue-green fluorescent bacteria in their rhizo- by presumably harmless ones (Penicillium,spheres, and suggested that the resistance of Mucor, actinomycetes and yeasts). Steamingthe cotton to root rot reflected antibiotic protec- reduced bacterial and actinomycete numbers intion at the high carbohydrate level. In further soil very markedly for about three months, yetwork, they demonstrated that the immunity of the number on the healthy roots of tomatoesmaize plants to Phymatotrichum root rot is was as high as on those of untreated soil, implyingattributable to protection afforded by its root- a protective and ameliorating influence of thesurface microflora. plant on the soil microflora in the rhizosphere.

Since soil treatment exerts marked effects on Katznelson (84) in general obtained results verythe soil microflora, the question naturally arises similar to those of West and Hildebrand (29),as to whether similar effects may be expected in particularly with fungi. That dried blood addedthe rhizospheres. Katznelson (84) obtained defi- to root rot soil induced healthy root formationnite evidence of stimulation of certain groups of was of particular interest.Organisms in the rhizosphere of mangels growing Zukovskaya (92a), working with potatoes,in manured soil. By means of the qualitative- flax and clover, found that the microbial popula-nutritional approach, Hildebrand and West (62) tion of the rhizosphere was 100 times as greatworking with strawberries showed that the as in the soil away from the root. Changes ofincorporation of soybean and red clover into the this microflora coincided with the development ofsterilized soil altered the bacterial equilibrium the plant. She also claimed that each plant, grownin both soil and rhizosphere so that untreated in the same soil, enhanced the activity of a spe-root rot soil gave bacterial balance index2 values cific microflora of its own.

2 The equilibrium between presumably "harm- ring rhizosphere types is designated as theful" bacteria and the innocuous, normally occur- bacterial balance index.


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142 J. H. STALLINGS [voL. 18

Using flax, Berezova (93) concluded that differ- cide applied as a spray, a seed treatment,ent types of organisms were associated with or a soil treatment. J. Econ. Entomol.,plants at different stages of growth. Some were 45, 302-7.found only during certain stages of development, 2. BRIAN, P. W., WRIGHT, JOYCE M., STUBBS,J., AND WAY, AUDREY M. 1951 Uptakesome during all stages, and others were present of antibiotic metabolites of soil micro-only at maturity. X organisms by plants. Nature, 167, 347-

DISEASERESISTANCE ~~~349.DISEASE RESISTANCE 3. MITCHELL, J. W., ZAUMEYER, W. J., ANDIt has been known for many years that some ANDERSON, W. P. 1952 Translocation of

varieties of plants are resistant to certain diseases streptomycin in bean plants and its effectwhile others grown under identical conditions on bacterial blights. Science, 115, 114-are not. This resistance factor is traceable to . N Nspeifigeetichraceritic (4, 9-10). 4. NELSON, C. I. 1952 The microorga ismsspecific genetic characteristics (46, 94-102). of our soil-Balance in soil life forms the

Plant breeders use this knowledge in breeding role of the actinomycetes. North Dakotaresistance into new varieties. More recently the Agr. Expt. Sta. Bimon. Bull., 15, 59-60.same situation has been found to be true with 5. Locxi AD, A. G., AND LANDERKIN, G. B.certain insects (103). 1949 Aspects of antagonisms in soil.Thus far no satisfactory explanation has been Plant and Soil, 1, 271-276.

advanced as to just how a specific gene, a gene 6. AHMAD, K., BUMPUS, F. M., DUNSHEE, B.pair, or combination of genes actually imparts R., AND STRONG, F. J. 1949 Antimycinresistance to either disease or insect. Apparently antibiotics. Federation Proc., 8, 178.it has been assumed that this influence is exerted 7. ARNSTEIN, H. R., COOK, A. H., AND LACEY,MARGARET S. 1948 The inhibition ofwithin the plant. Recent discoveries in other, Fusarium oxysporum var. cubenee bybut related fields, indicate that this influence may musarin, an antibiotic produced by Mere-be exerted by influencing the microflora of the dith's actinomycete. J. Gen. Microbiol.,rhizosphere. These findings indicate this genetic 2, 111-122.influence may determine the quality of substances 8. BAWDEN, F. C., AND FREEMAN, G. G. 1952excreted or sloughed off by the growing roots. The nature and behaviour of inhibitors ofThese products in turn determine the composition plant viruses produced by Trichotheciumof the microflora of the rhizosphere. roseum Link. J. Gen. Microbiol., 7, 154-Walker and collaborators (104) showed that 168.

.brw-kne.noaie 9. DuBos, R. J., AND HOTCHXSS, R. D. 1941certain brown-skmined onion varieties are re- The production of bactericidal substancessistant to smudge (Colletotrichum citcinane) be- by aerobic sporulating bacilli. J. Exptl.cause of the presence of protocatechuic acid and Med., 73, 62-640.catechol in the tissue. Greathouse and Rigler 10. FORSTER, R. 1950 Inactivation of the(105, 106) also demonstrated this principle in virus of tobacco mosaic by filtrates fromtheir characteristic series of Phymatotrichum Trichoderma. Bragantia, 10, 139-148.omnivorum to different phenolic compounds and 11. SLAGLE, C. W., WOwYRZ, STLVIA, ANDalkaloids. According to Johnson and Schaal PRICE, W. C. 1952 Inhibition of plant(107) resistance to common scab (Streptomyce8 virus infection by growth products ofscabies) of potato varieties is related closely to Neurompora. Phytopathology, 42, 240-the presence in the superficial layer, especially in 12.WA4SMAN,S. A., AND WOODRUFF, H. B.and near the lenticels, of chlorogenic acid, a 1942 The occurrence of bacteriostaticphenolic compound, and possibly also of tyro- and bactericidal substances in the soil.sinase. Chlorogenic acid or its quinone also Soil Sci., 53, 233-239.accumulates in the periderm and around injuries 13. NICKELL, L. G., AND BURKHOLDER, P. R.whether mechanical or parasitic in origin. 1947 Inhibition of Azotobacter by soil

actinomycetes. J. Am. Soc. Agron., 39,REFERENCES 771-779.

14. KIDO, G. S., AND SPYHALSKI, E. 19501. ASHDOwN, D., AND CORDNER, H. B. 1952 Antimycin A, an antibiotic with insec-Some effects on insects and insect control ticidal and miticidal properties. Science,and plant response of a systemic insecti- 112, 172-173.


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15. MERTIN, NonxW, AND GOTTLIUB, D. 1952 ta cover crops. Can. J. Research, C,The production and role of antibiotics in 19, 199-210.the soil: III. Terramycin and aureomycin. 30. WAKSMAN, S. A., BuGiz, EIIZABnrrH, ANDPhytopathology, 42, 294-296. RHILzY, H. CHRISTINE 1944 Bacterio-

16. SImNO"F, P., AND GoTIuzB, D. 1951 static and bacteriocidal properties ofThe production and role of antibiotics in antibiotic substances with special refer-the soil: I. The fate of streptomycin. ence to plant pathogenic bacteria. Bull.Phytopathology, 41, 42030. Torrey Botan. Club, 71, 107-121.

17. WEINDLNG, R. 1934 Studies on a lethal 31. WAxsMAN, S. A., AND HORNING, ELIZABETHprinciple effective in the parasitic action 5. 1943 Distribution of antagonisticof Trichoderma lignorum on Rhizoctonia fungi in nature and their antibiotic ac-solani and other soil fungi. Phytopa- tion. Mycologia, 35, 47-65.thology, 24, 1153-1174. 32. HzssAYON, D. G. 1953 Fungitoxins in the

18. AlaN, M. C., AND HAUNSELER, C. M. 195 soil: II. Trichothecin, its production andAntagonistic action of Trichoderma on inactivation in unsterilized soils. SoilRhizoctonia and other soil fungi. Phyto- Sci., 75, 395-404.pathology, 25, 244-252. 33. JEFFEyS, E. C. 1952 The stability of

19. ANWAR, A. A. 1949 Factors affecting the antibiotics in soils. J. Gen. Microbiol.,survival of Helminthosporium sativun and 7, 295-312.Fusarium lini in soil. Phytopathology, 34. GOTTLIEB, D., SIMINOFF, P., AND MARTIN,39, 1005-1019. MARY M. 1952 The production and role

20. JOHNSON, D. E. 1931 The antibiosis of of antibiotics in the soil: IV. Actidionecertain bacteria to smuts and some other and clavacin. Phytopathology, 42, 493-fungi. Phytopathology, 21, 843-3. 496.

21. BIGGER, J. W. 1950 Synergism and antag- 35. RHIDE, V. P., MONIZ, L., AND PATIL, R. B.onism as displayed by certain antibac- 1952 Actinomycetes antibiotic to plant-terial substances. Lancet, 259, 46-50. pathogenic bacteria. Current Sci. (In-

22. SPENCER, SOPHIE 1950 Bacteriologic studies dia)-, 21, 70-71.of the newer antibiotics-Effect of com- 36. ZORIL, J. G. 1952 Microorganisms antag-bined drugs on microorganisms. J. Lab. onistic to certain fusaria. J. Colo.-Wyo.Clin. Med., 86, 183-191. Acad. Sci., 4, 62.

23. MEREDITH, C.- H., AND SEMENIUK, G. 1946 37. LEBEN, C., ARNY, D. C., AND KzEITT, G. W.The antagonism of some species of actino- 1953 Small grain seed treatment with themycetes in relation to soil-inhabiting antibiotic, helixin B. Phytopathology,plant pathogens. Iowa Agr. Expt. Sta. 48, 391-394.Ann. Rept. 1945-1947, 199-202. 38. ZAuMETER, W. J. 1953 Alfalfa yellow

24. GILLIVER, K. 1946 The inhibitory action mosaic virus systemically infectious toof antibiotics on plant pathogenic bacteria beans. Phytopathology, 48, 38-42.and fungi. Ann. Botany, 10, 271-282. 39. ANSLOW, W. K., RAISTRICK, H., AND SMTH,

25. YOUNG, H. C., AND BRANDT, W. H. 1953 G. 1943 Antifungal substances fromTimber decay and deterioration observed moulds: Part I. Patulin (anhydro-3-in oak wilt experiments. Ohio Agr. Expt. hydroxymethylene - tetrahydro 1 -:4 -

Sta. Farm and Home Res., 38, 8-9. pyrone-2-carboxylic acid), a metabolic26. GOTTLIEB, D., AND SIMNOFF, P. 1952 The product of Penicillium patulum bainier

production and role of antibiotics in the and Penicillium expansum (Link) Thom.soil: II. Chloromycetin. Phytopathology, J. Soc. Chem. Ind., 62, 236-238.4, 91-97. 40. WHIFFEN, ALmL& A. 1948 The production,

27. WAESMAN, S. A. 1947 Microbial antag- assay, and antibiotic activity of actidione,onisms and antibiotic substances. The an antibiotic from Streptomyces griseu.Commonwealth Fund, New York, N. Y. J. Bacteriol., 56, 283-291.

28. BROWN, J. G., AND BowLE, AUicz M. 1944 41. GROsSBARD, E. 1948 Investigations on28.~~ ~BRW'.GADBoE LC .14 microbial antagtonisms and antibiotic sub-Effect of penicillin on a plant pathogen. saces. antagois. Sta. o(chsuntPhytopathology, 34, 760-761. Herts), 34th Ann. Rept.37-(Cheshunt4

29. WEST, P. M., AND HILDBRAND, A. A. 1941 2 BROWN,J. G., AND BoLt, ALICE M. 1944The microbiological balance of strawberry Penicillin treatment of crown gall. Sci-root rot in soil as related to the rhizo- ence, 100, 528.sphere and decomposition effects of cer- 43. LEBEN, C., AND KEITr, G. W. 1948 An


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antibiotic substance active against certain 57. RAPER, K. B., AND THOM, C. 1949 Aphytopathogens. Phytopathology, 38, manual of the Penicillia. The Williams899-906. and Wilkins Co., Baltimore, Md.

44. HESSAYON, D. G. 1951 'Double-action' of 58. CROWDY, S. H., AND WAIN, R. L. 1950trichothecin and its production in soil. Aryloxyaliphatic acids as systemic fungi-Nature, 168, 998-999. cides. Nature, 165, 937-938.

45. RISBETH, J. 1950 Observations on the 59. McNEw, G. L., AND SUNDHOLM, K. 1949biology of Fomes annotue with particular The fungicidal activity of substitutedreference to East Anglian pine plantations: pyrozoles and related compounds. Phyto-I. The outbreaks of disease and ecological pathology, 39, 721-751.status of the fungus. Ann. Botany, 14, 60. HORSFALL, J. G., AND DIMOND, A. E. 1951365-383. Plant themotherapy. Ann. Rev. Micro-

46. COOPER, S. H., AND CHILTON, S. J. P. 1948 biol., 5, 209-222.Antibiosis and sugar cane root rot. 61. ZENTMEER, G. A., HORSFALL, J. G., ANDPhytopathology, 38, 6. WALLACE, P. P. 1946 Dutch elm disease

47. STESSEL, G. J., LEBEN, C., AND KEiTT, G. and its chemotherapy. Conn. Agr. Expt.W. 1953 Screening tests designed to dis- Sta. Bull. 498.cover antibiotics suitable for plant disease 62. HILDEBRAND, A. A., AND WEST, P. M. 1941control. Mycologia, 45, 325-334. Strawberry root rot in relation to micro-

48. GREGORY, K. F., ALLEN, 0. N., RIKER, A. biological changes induced in root rotJ., AND PETERSON, W. H. 1952 Anti- soil by the incorporation of certain coverbiotics as agents for the control of certain crops. Can. J. Research, C, 19, 183-198.damping-off fungi. Am. J. Botany, 39, 63. GROSSBARD, E. 1947 The control of plant405-415. diseases by microbial antagonsim. Expt.

49. WHIFFEN, ALMA A., BOHONOS, N., AND Res. Sta. (Cheshunt, Herts.), 33rd Ann.EMERSON, R. L. 1946 The production of Rept., 29-39.an antifungal antibiotic by Streptomyces 64. CLARK, F. E. 1940 Notes on types of bac-griseus. J. Bacteriol., 52, 610-611. teria associated with plant roots. Trans.

50. LECHEVALIER, H., ACKER, R. F., CORKE, C. Kansas Acad. Sci., 43, 75-84.T., HAENSELER, C. M., AND WAKSMAN, S. 65. BAWDEN, F. C., AND PIRIE, N. W. 1936A. 1953 Candicidin, a new antifungal Experiments on the chemical behavior ofantibiotic. Mycologia, 45, 155-171. potato virus "X". Brit. J. Exptl. Pathol.,

51. SANFORD, G: B., AND BRADFOOT, W. C. 17, 64-74.1931 Studies of the effects of other soil- 66. STANLEY, W. M. 1934 Chemical studies oninhabiting microorganisms on the viru- the virus of tobacco mosaic: I. Some ef-lence of Ophiobolus graminis Sacc. Sci. fects of trypsin. Phytopathology, 24,Agr., 11, 512-528. 1055-1085.

52. LUIJK, A. VAN 1938 Antagonisms between 67. SILL, W. H., JR., AND WALKER, J. C. 1952various microorganisms and different A virus inhibitor in cucumber in relationspecies of the genus Pythium parasitizing to mosaic resistance. Phytopathology,upon grasses and lucerne. Meded. Phyto- 42, 349-352.path., Lab. Willie Commelin Scholten, 68. CASIDA, J. E. 1942 The role of plants inBaarn, 14, 43-83. the production of systemic insecticides.

53. GIBSON, T. 1949 The present state of Com. Fertilizer, 85, 48.development of soil bacteriology. Agr. 69. CHAO-SENG, T. 1950 Protection againstProgr., 24, 108-111. aphids by seed treatment. Nature, 166,

54. EATON, F. M., AND RIGLER, N. E. 1946 909-910.Influence of carbohydrate levels and root- 70. Ivy, E. E. 1952 Testing systemic insec-surface microfloras on Phymatotrichum ticides. Agr. Chemicals, 7, 44-45, 127.root rot in cotton and maize plants. J. 71. JOHNSON, J. 1941 Chemical inactivationAgr. Research, 72, 137-161. and the reactivation of a plant virus.

55. VANTERPOOL, T. C. 1952 The phenomenal Phytopathology, 31, 679-701.decline of Browning root rot (Pythiumr 72. RIPPER, W. E., GREENSLADE, R. M., ANDspp.) on the Canadian prairies. Sci. Agr., HARTLEY, G. S. 1950 A new systemic32, 443-452. insecticide bis (bis dimethylamino phos-

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