J, Phytopathology 126, 149—159 (1989) © 1989 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1785 Instituto Biologico, Sao Paulo, Brasil Bacillus thuringiensis — A New Perspective for an Induced Protection to Coffee Leaf Rust* DAGMAR S. RovERATn*'% ANA REGINA R. TEIXEIRA and WALKYRIA B. C. MORAES Authors' address: Se^ao de Bioquimica Fitopatologica, Instituto Biologico, CP. 7119, 0105! Sao Paulo, SP. (Brasii). With one figure Received December 14, 1988; accepted January 30, 1989 Abstract Susceptible coffee plants {Coffea arahica cv. 'Mundo Novo') treated with a suspension of Bacillus thuringiensis or with three of commercial products (Thuricide HD, Bactimos and Bactospeine PM) of B. thuringiensis were protected against a later inoculation with Hemilem vastatrix, the causal agent of coffee leaf rust. Thuricide HD was effective at concentrations from 5 to 20 mg/ml; at this concentration, protection reached 90 %, lasted for at least 5 weeks, and was systemic. Induced resistance was determined by the reduction of the average number of lesions per leaf iti treated leaves when compared with non-treated controls. A decrease in lesion size as well as a delay in sporulation time were also observed in protected plants treated with Thuricide HD. Zusammenfassung Bacillus thuringiensis — eine neue Perspektive fiir den induzierten Schutz gegenuber Kaffee-Blattrost Anfallige KaffeepOanzen (Coffea arahica cv. 'Mundo Novo'), die entweder mit einer Suspen- sion von Bacillus thuringiensis oder mit drei kommerzielJen 3. thuringiensis-Produkten (Thuricide HD, Bactimos und Bactospeine PM) behandelt worden waren, wurden so gegen eine spatere Inokulation mit Hemileia vastatrix, dem Erreger von Kaffee-Biattrost, geschiitzt. Thuricide HD bewirkte bei Konzentrationen von 5—20 mg/ml einen 90%igen Schutz, der mindestens 5 Wochen andauene und systemisch war. Diese induziene Resistenz liefi sich bestimmen durch die Reduzierung '^ Research supported by the grant PN 79.2186.9.01.200 from the "Deutsche Gesellschaft fur Technische Zusammenarbeit GmbH". ''"^ Fellow of Brazilian National Research Council, CNPq. U.S. CQpyrigh<Cfc™.ceC=nwr Code Su«n.™; 0931-1785/89/2602-0149$02.50/0 150 DAGMAR S. ROVERATH, ANA REGIKA R. TEIXEIRA and WALKYRIA B. C. MORAES der durchschnittlichen Zahl von Lasionen pro Blatt bei behandelten Blauern im Vergleich zu unbehandelten Kontrollen. Es liefien sich in den Pflanzen, die mit Thuricide HD behandelt worden waren, sowohl eine Abnahme der Lasionengrofie als auch eine Zeitverzogerung in der Sporenbildung feststellen. Resumo Cafeeiros suscetiveis {Coffea arabica cv. 'Mundo Novo') previamente tratados com Bacillus thuringiensis ou com produtos comerciais a base deste microorganismo (Thuricide HD, Bactimos e Bactospeine PM), apresentaram-se protegidos contra uma posterior lnfec^ao com o fungo Hemileia vastatrix, agente causa) da ferrugem alaranjada do cafeeiro. O Thuricide HD, foi efetivo em induzir prote^ao nas plantas desde a concentra^ao de 5 mg/ml, sendo que o maximo de prote^ao foi atingido quando se empregou concentra^oes de 20 mg/ml. Nesta concentrai;ao, a protegao alcangada ficou em tomo de 90 % e perdurou por 5 semanas. A prote9ao observada foi sistemica. O indice de prote^ao foi determinado peia redugao do numerc medio de lesoes por folha. Uma redu^ao no tamanho das lesoes nas folhas tratadas, bem como um aumento do periodo de latencia do patogeno e o consequente retardamento de sua esporulajao tambem foram observados. Coffee is one of the most important crops in Brazil and its production is highly affected by coffee leaf rust, a disease caused by the obligate fungus Hemileia vastatrix Berk, et Br. The disease is currently controlled by copper fungicides, which significantly increases management costs {BERGAMIN and PAIVA 1983). The high costs of fungicides, the consequent pollution of the environment, and the risks of an increase in pathogen resistance to pesticides and a predisposi- tion to infection by other pathogens, have led plant pathologists to intensify the search for alternative controls. Several authors have found that plants can be induced to be resistant against infectious agents when properly stimulated (Kuc 1985, DEAN and Kuc 1985, ScHOENBECK et al. 1980), and that plants may be protected either locally or systemicaliy (TUZUN and KuC 1985, DE WIT 1986}. Induced resistance has been achieved in many plant-pathogenic systems by treatment with non-pathogenic microorganisms (SCHOENBECK et al. 1980), aviru- lent races of the pathogen (OUCHI et al. 1974), inactivated pathogens {BELL and PRESLEY 1969, JENNS and Kuc 1980), and metabolites released hy microorganisms (GRAHAM et al. 1977, Guzzo et al. 1987). The induced protection was shown to be effective under field conditions (BALDER and SCHOENBECK 1983, CARUSO and Kuc 1977), thus opening new perspectives for plant disease control that may reduce the use of chemical pesticides. Biological control of plant diseases (PUSEY and WILSON 1984, BAKER et al. 1985) and pests (DEACON 1983) has been demonstrated previously using non- pathogenic bacteria. One of these, the enthomopathogen — Bacillus thuringien- sis, used for pest control, has already been used in field experiments and commercialized as a biologic insecticide (FIGUEIREDO et al. 1960, 1961, LuTHY et al. 1982). As we have demonstrated previously that challenge inoculation of coffee plants with heat-killed urediniospores of H. vastatrix, non-pathogenic fungi, or bacteria (MARTINS et al. 1985), protects them systemically against the pathogen (MORAES et al. 1976, BERETTA et al. \^77), we have evaluated the potential Bacillus thuringiensis — A New Perspective 151 effectiveness of B. thuringiensis as an inducer of resistance against H. vastatrix, under greenhouse condition, in order to find an alternative method for coffee leaf rust control, which could be used in the field and associated low costs, low environmental impact and a possible control of coffee pests. Materials and Methods Plant and fungal materials Coffee plants [Coffea arabica L. cv. 'Mundo Novo') were grown from seeds, which were sown in sand inside shaded boxes (1.20 x 0.80 X 1.0 cm) and watered every other day with a volume sufficient to maintain 95 % relative humidity in the box. Seedlings were transferred to a composted soil in plastic bags (10 cm diam.) when the cotyledonary leaves had developed. Plants were then grown in the greenhouse under prevailing conditions (21—25 "C, 12 h light daily). Every other week, plants received a nutrient solution containing approximately 2.5 ppm of nitrogen. Plants were used when they had developed 8—12 pairs of fully expanded leaves (approximately 12—18 months old). Urediniospores of the pathogen, Hemileia vastatrix Berk, et Br. predominantly race II, were obtained from young lesions of naturally infected coffee plants hy gently brushing from the leaves. The urediniospores were passed through a 100-mesh sieve and kept in cryotuhes in liquid nitrogen until used. Inducer preparations The following commercial products having B. thuringiensis Berliner as the active component were used in the biological assays to induce resistance in coffee plants against the pathogen: Thuricide HD {B. thuringiensis var. kurstaki, 16,000 UI); Bactimos (fi. thuringiensis var. israelensis, 6,000 UI); Bactospeine PM (B. thunngiensis var. kurstaki, 16,000 UI); B. thuringiensis var. israelensis in liquid formulation (1,000 UI); B. thuringtensis var. kurstaki (17,000 UI); B. thunngiensis var. kurstaki lyophilized (80,000 UI), Thuricide HD was obtained from Sandoz do Brasil whereas all the other preparations were from Salsbury Laboratory, Brazil. All the commercial products were suspended in sterile distilled water at different concentrations and stjrred for 10 min before application to plants. An isolate of B. thuringiensis var, kurstaki obtained from the Genetics Department of the Agriculture School "Luiz de Queiroz", Piracicaba, S.P., Brasil, was also used as inducer. Cultures of B. thuringiensis were maintained in 125 ml flasks containing liquid medium (PBES) prepared with 0.8 % (w/v) peptone, 0.8 % (w/v) beef extract, 0.5 % (w/v) sodium chloride in sterile distilled water (pH 6.1), without shaking, at 37 "C in the dark, and were sub-cultured every 8 days. These cultures were referred to as "mother-cell" suspensions. Suspensions of B. thuringiensis were prepared in PBES by diluting I.O ml of the "mother-cell" suspension into 75 ml of PBES in a 250 mi flask. After two days m the dark at 37 °C without shaking, 1.0 ml ahquots of the cell suspension were transferred to PBES solidified with 1.5 % agar in Petri dishes. The plates were kept for 2 days in the dark at 37 °C. Cells were washed off the agar surface with 10 ml of sterile distilled water, and the suspension was adjusted to a concentration of approximately 4.0 X 10* cells per ml. This initial suspension was diluted With water \ : 2 and 1 :4, and the three preparations were used as inducers. Inoculum preparation Cryotubes containing urediniospores were taken from liquid nitrogen, heat shocked for 10 min at 40 °C in a waterbath, and distilled water was added to a concentration of 2 mg/mi. The suspension was then stirred for 10 min with a magnetic sdrrer, and sonicated for 30 sec (50 Hz). The suspension was constantly stirred during the inoculation process outlined below. Biological assays Groups of 10 coffee plants were used for each treatment within biological assays. Unless otherwise specified, second, third and fourth pairs of leaves from the top to the bottom were selected, washed with tap water, gently dried with cotton and sprayed on the abaxial (under) leaf surface with 152 DAGMAR S. ROVERATTl, ANA REGINA R. TEIXEIRA and WALKYRIA B. C. MORAES the inducer preparation, using nitrogen as the propellent gas, at a constant pressure of 2.5 bars. Controls received water instead of the inducer. Plants were maintained in the greenhouse. Seventy- two hours later, the treated and control plants were inoculated with the pathogen (2 mg/ml in distilled water) by spraying the urediniospore suspension onto the abaxial surface of the same previously treated leaves. Inoculated plants were maintained in conirolied chambers for 48 h at 24 °C and 90 % RH in the dark. After that, the chamber was adjusted to a 12 h photoperiod of 5,000 lux, and plants were kept at this condition until the sytnpioms appeared, 25—35 days later. The percentage of protection (% P) was calculated according to MORAES et al. (1976). In some cases, the type of lesions that developed was also considered as well as the interval of time for sporulation. Systemic protection assays The systemic effea of the inducer was studied only for the commercial product Thuricide HD. For this, aqueous preparations of Thuricide HD at a concentration of 20 mg/ml were sprayed as previously described in groups of ten piants each, according to the following schedules. (1) The third pairs of leaves were treated with the inducer. Seventy-rwo hours laier, tbe treated leaves were excised and the second and fourth pairs of leaves were inoculated with the pathogen as previously described; (2) only the third pairs of leaves were treated with the inducer, and 72 h later the untreated leaves immediately adjacent to the treated ones were inoculated; (3) inducer preparation was sprayed onto the adaxial (upper) leaf surface of the second, third and fourth pairs of leaves at 72 h before inoculation of the pathogen onto the abaxial surface of the same treated leaves; (4) groups of 15 plants each had one leaf" of the third pair treated with the inducer preparation at 72 h before both leaves of the same pair were inoculated. Controls for each group received sterile distilled water instead of the inducer before the inoculation. Besides the percentage of protection, the different types of lesions were also considered for the evaluation of symptoms in systemically induced plants. They were classified according to the size of the lesions and time of sporulation, as the following: Type I; small chlorotic points (less than 1 mm diameter). Sporuiation occurred 42 days after the inoculation. Type II: small round chlorotic lesions (between 1 and 2 mm). Sporuiation occurred 35 days after inoculation. Type III: coalescent chlorotic lesions (larger than 2 mm). Sporulation took place 27 days after inoculation. Statistical analysis Experiments were replicated at least three times. Results from the biological assays were statistically analyzed. Differences between means were tested for significance according to Student's paired t-test, at P :< 0.05. Table 1 Protection of coffee plants against a cultivar pathogenic race of H. vastatrix by prior treatment of leaves with an aqueous suspension of Thuricide HD Protection (% of control) 69.1 69.1 %.4 98.8 97.6 Inducer applied on the abaxial surface of 2nd, 3rd and 4th pairs of leaves 72 h before the inoculation with a suspension of urediniospores of H. vastatrix (2 mg/ml). Controls received distilled water instead of the inducer. Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the control (P ^ 0.05) according to the Student's t-test. Inducer concentration (mg/mi)' 5 10 20 50 100 Control Average No. lesions/ieaf 2.6 ± 0.7* 2.6 ± 0.5* 0.3 ± 0.1* 0.1 ± 0.0* 0.2 ± 0.0'^ 8.4 ± 1.4 Bacillus thuringiensis — A New Perspective 153 Results Effect of different concentrations and time-course of protection of Thuricide HD As shown in Table 1, all concentrations of the commercial product Thuri- cide HD protected coffee plants against infection by the pathogen, and optimal results were achieved at an inducer concentration of 20 mg/ml. At this concentra- tion, protection lasted for at least 5 weeks (Tables 1, 2). Table 2 Influence of time intervals in days or weeks between the application of Thuricide EiD and inoculation with a cultivar-pathogenic race on the induced protection of coffee plants Time interval in days' 2 4 6 8 10 12 14 16 Control in weeks' I 2 3 4 5 Control Average No. lesions/ieaP 0.9 ± 0.2-'' 1,1 ± 0.2* 0.4 ± 0.1* 1.5 ± 0.3* 0.9 ± 0.2* 1.3 ± 0.3* 1.1 ± 0.2''' 1.5 + 0,4'^ 11.4 ±2.8 • 1.3 ± 0.5' 0.3 ± 0.1*^ 0.7 ± 0.2* 0.8 ± 0,2* 1.9 ± 0.6* 10.6 ± 3.3 Protection (% of control) 92.1 90.4 96.5 86.8 92.1 88.6 90.4 86.8 — 87.7 97.2 93.4 92.5 82.1 — ' Inducer applied on the abaxial surface of 2nd, 3rd and 4th pairs of leaves at defined intervals in hours or weeks before inoculation with a suspension of urediniospores of//. vastatrix (2 mg/ml in distilled water). Controls received water instead of the inducer. ^ Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the contra! (P < 0.05) according to the Student's t-test. Evidence that the protection by Thuricide HD is systemically induced When applied at a distance from the inoculation site Thuricide HD effec- tively protected plants from H. vastatrix infection regardless the initial site of Thuricide treatment (Tables 3 and 4). Systemic protection was observed in both proximal and distal directions relative to the position of the induced leaf (Table 3). The systemic effect also spread laterally from one leaf of the pair to the other leaf of the same pair (Table 3), and in the same leaf from the adaxial to the abaxial surface (Table 3). Even after excision of the treated leaves, the systemic effect could be observed in the remaining leaves (Table 4). 154 DAGMAR S. ROVERATTI, ANA REGINA R. TEIXEIRA and WALKYJUA B. C. MORAES Tahle 3 Local and systemic effect of Thuricide HD as an inducer of protection of coffee plants against a cultivar-pathogenic race of H. vastatrix Leaf pair Average No. Protection Type of lesions' Treatment position lesions/leaf"" (% of control) I 11 III Untreated 2nd 39.9 ± 6.9" 64.3 + -\-+ - Treated leaf 3rd 51.8 111.0* 71.3 + ++ - Untreated 4th 71.0 ± 12.9' 63.2 + ++ - Control 2nd 111.9 123.9 — - - 3rd 180.5 1 33.6 — - - 4th 192.9 + 43 1 — - - Treated leaf 3rd 27.1+ 5.3'^ 85.0 + ++ - Opposite leaf 3rd 66.t 1 9.7'^ 63.4 + ++ - Control 3rd 180.5 1 33.6 — - - + + + Treated on 2nd 40.2 1 12.0* 64.1 + + + the opposite 3rd 47.6 1 15.3* 73.6 + ++ - side of leap 4th 56.6 + 8.7* 70.7 4- ++ - Control 2nd 111.9 ±23.9 — - - + + + 3rd 180.5 ± 33.6 — - - + + + 4th 192.9 1 43.1 — - - + + -h ' Third pairs of leaves were treated with a suspension of Thuricide HD (20 mg/ml) 72 h before the inoculation of the 2nd, 3rd and 4th pairs of leaves with an aqueous suspension of urediniospores of H. vastatrix (2 mg/ml). Controls received water instead of the inducer. ' Only one leaf of the 3rd pair treated with a suspension of Thuricide HD (20 mg/m!) 72 h before the inoculation of the opposite leaf, which received only water instead of the inducer, and the induced leaf with an aqueous suspension of urediniospores of H. vastatrix (2 mg/ml). ' All the three pairs of leaves were treated on the adaxial leaf surface with a suspension of Thuricide HD (20 mg/m!) 72 h before the inoculation of the opposite leaf sides (abaxial) with an aqueous suspension of urediniospores of H. vastatrix (2 mg/ml). Controls received water instead of tbe inducer according to the same schedule as for treatments. •" Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the controls {P < 0.05) according to Student's t-test, * Lesion types as described in text, -f (present), - (absent). Effect of other commercial products and a cell suspension of B. thuringiensis Protection against infection by the coffee leaf rust fungus was observed when other commercial products were used (Table 5). Importantly, similar results were obtained when coffee plants were treated with a cell suspension of B. thuringiensis (Table 6) demonstrating that induced protection was a property of the bacterium and not a component of the commercial preparations. Effect of treatment on the type of lesions Regarding the type of lesions present in locally or systemically protected leaves, it was shown that besides being significantly fewer in number, the lesions were smaller in size when compared with those in control plants. In induced Bacillus thuringiensis — A New Perspective 155 Table 4 Persistence of the systemic effect of Thuricide HD after the excision of treaied leaves Leaf pair Average No. Protection Type of lesions' Treatment' position lesions/leaF (% of control) I II III Treaied 2nd Treated 4dl Gontroi 2nd Gontroi 4th ' Third pair of leaves treated with a suspension of Thuricide HD (20 mg/ml). Seventy-two hours later the treaied leaves were excised and the second and fourth pairs were inoculated with a suspension of urediniospores of H. vastatrix (2 mg/ml), Gontrols received distilled water instead of the inducer. - Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the controls (P < 0.05) according to Student's t-test, ' Lesion cypes as described in text, + (present), - (absent). 42,9 ± 14 74.5 ± 20 152.0 ± 22 135.0 ± 26 .7^- .8 .0 71.8 44,8 — Fig. L Different lesion types on coffee leaves inoculated with H. vastatrix. (a) Type III, large (> 2 mm) sporulating lesions characteristic of untreated control plants; (b) Type II, small (1—2 mm) chlorotic lesions that occur on plants induced to exhibit resistance to H. vastatrix by pretreatment with Thuricide HD; (c) Type I, small (< 1 mm) chlorotic lesions on leaves induced to exhibit resistance to H. vastatrix by pretreatment with Thuricide HD plants, sporulation was also shown to be delayed in comparison with the time required for fungal sporulation in controls (Tables 3, 4 and Fig. 1). Discussion Treatment of coffee leaves with commercial products containing B. thurin- giensis or with a pure suspension of the bacterium was shown to induce local protection against a later inoculation with a cultivar-pathogenic race of H. vasta- 156 DAGMAR S. ROVERATD, ANA REGINA R, TEIXEIRA and WALKYRIA B. C, MORAES Table i Protective effect induced in coffee plants by commercial products having B. thuringiensis as the tnain component Treatment' Thuricide HD Bactimos Bactimos Bactospeine PM Bactospeine PM B. thuringiensis israelensis liquid B. thuringiensis kurstaki liquid B. thuringiensis lyophilized B. thuringiensis lyophilized Control Concentraiion (mg/ml) 20 20 60 10 20 40%' 2%^ 10 20 — Average No. lesions/leaP 2.7 ± 0.7* 8.3 ± 2.9* 0.9 ± 0.3* 2.9 ± 0.6* 1.3 ± 0.2* 1.1 ± 1.1* 4.9 ± 0.9* 0.3 ± 0.1* 0.3 ± O.l-^ 11,5 ± 2,7 Protection (% of control) 76.5 27.8 92^ 74.8 88.7 90.4 57.4 97.4 97.4 — Second, third and fourth pairs of leaves treated with an aqueous suspension of commercial products at the indicated concentrations, 72 h before inoculation with a suspension of urediniospores of H. vastatrix (2 mg/ml). Controls received distilled water instead of the inducer. Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the control (P < 0.05) according to the Student's t-test. Commercial liquid preparations of the two inducers were diluted with distilled water to the concentration given in percentage of the initial ones. Tahle 6 Protection of coffee plants against a cultivar-pathogenic race of H. vastatrix by pHor treatment of leaves with an aqueous suspension of B. thuringiensis var. kurstaki grown in culture medium B. thuringiensis concentration Average No. Protection No. of ceils/ml' lesions/leaF (% of control) 4.4 X 10' 6.4 ± 2.8* 83.8 2.2 X 10* 6.3 ± 2.4* 84.1 1,1 X 10* 14,1 ± 4.3* 64J Control 39,5 ± 14.7 — Second, third and founh pairs of leaves treated with an aqueous suspension of living cells of B. thuringiensis kurstaki grown in sohd medium, 72 h before inoculation with a suspension of urediniospores of H. vastatrix (2 mg/ml). Controls received distilled water instead of the inducer. Values (± standard error) are means of three replicates. Means followed by an asterisk are significantly different from the control (P ^ 0.05) according to the Student's t-test. Bacillus thuringiensis — A New Perspective 157 trix. At concentrations up to 20 mg/ml, the local protection caused by Bactimos, Bactospeine PM, Thuricide HD, commercial liophylized cells of B. thuringiensis and the cell suspension of the bacillus grown in culture medium was between 80 and 95 %. Biological assays with Thuricide HD showed that this product was very active as an inducer of resistance even in concentrations lower than 20 mg/ ml, and the induced resistance lasted for at least 5 weeks. Other authors have demonstrated similar results in other plant-pathogen systems. For example, Kuc and RICHMOND (1977) working with cucumbers, showed that inoculation of the first true leaf with Colletotnchum lagenarium when the second leaves were not completely expanded, systemically protected the plants against a later inoculation with the same pathogen for 4—5 weeks. A second inoculation undertaken 3 weeks later, extended the protection for a longer period. CLOUD and DEVERALL (1987) have shown that the development of anthrac- nose in trifoliate leaves of beans was significantly reduced when the primary leaves of the same plants had been inoculated first with the pathogen. DOKE et al. (1987) demonstrated that treatment of potato leaves with hyphal wall components of Phytophthora mfestans systemically protected the plants against a later inoculation with the same pathogen, while BERETTA et al. (1977) showed that coffee leaves treated with the water washes from autoclaved urediniospores of H. vastatrix induced a systemic protection against the pathogen. Different isolates of bacteria have been associated with the resistance induced in plants against pathogenic fungi. DEHNE et al. (1984) observed that treatment of wheat plants with B. suhtilis or its metabolic products induced a high level of resistance against Erysiphe graminis f.s. tritiei. Pathogen development was consid- erably inhibited, and there was a significant reduction in sporulation intensity and formation of cleistotecia. NELSON (1988) has also shown that thirteen strains of Enterohacter cloacae and Erwinia herhicola were able to reduce the incidence of Pythium seed rot and preemergence damping-off of cotton, by controlling the pathogen biologically. Thus, the evidence presented in this paper that cell suspensions of B. thuringiensis, or its commercial products, are able to protect coffee plants against the coffee leaf rust fungus are corroborated by the results from other authors for different plant-pathogen systems, using bacteria as inducers of resistance. According to Kuc! (1987), systemically induced resistance does not abso- lutely immunize the treated plants, Instead, as reported here for coffee, disease severity is significantly reduced, i.e. there is a decreased number of lesions and a longer time is required for sporulation to occur. Results from the present research as well as those from our previous studies (BERETTA et al. \977, MARTINS et al. 1985, 1986) suggest that the protection induced in coffee is a non-specific phenomenon which can be achieved by inducers of apparently different nature. This fact, associated with the persistence of the systemically-induced effect enhances the prospects for using microorgan- isms as an alternative method of control for coffee leaf rust in the field and for reducing the use of chemical fungicides. Experiments are being undertaken in the field to verify this possibility. That commercial formulations of B. thuringiensis 158 DAGMAR S. RovEKATn, ANA REGINA R. TEIXEIRA and WALKYRIA B. C. MORAES and suspensions of its cells were effective in inducing protection against coffee leaf rust, and this suggests that the costs of coffee production might be reduced significantly using B. thuringiensis in an integrated program for the control of insect pests and fungal diseases. The authors are indebted to Dr. M. B. FlGUElREDO frotn the Instituto Biologico, Sao Paulo, Brazil, to Dr. RjCHARD C. STAPLES from tbe Boyce Thompson Institute. N.Y., USA, and to Professor RALPH L, NICHOLSON from Purdue University, Ind,, USA for their assistance in the tnanuscript preparation and revision. Literature BAKER, C, J,, J. R. STAUELY, and N, MOCK, 1985: Biocontrol of bean rust by Baallus subtilis under field conditions. Plant Dis. fS, 770—772. BALDER, H., and F. SCHOENBECK, 1983: The efficiency of induced resistance under practical culture conditions. II. Rust of chrysanthemum and carnation, downy mildew of rape, cucumber and lettuce. J. Plant Dis. and Protection 90, 200—206. BELL, A. 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