Resistance induction in plants 547

RESISTANCE INDUCTION IN WHEAT PLANTS BY SILICON AND

Flávia Batista Gomes1; Jair Campos de Moraes2*; Custódio Donizete dos Santos3; Márcio Marcos Goussain1

1 UFLA - Programa de Pós-Graduação em Agronomia/Entomologia, C.P. 3037 - 37200-000 - Lavras, MG - Brasil. 2 UFLA - Depto. de Entomologia. 3 UFLA - Depto. de Química. *Corresponding author

ABSTRACT: The pest greenbug graminum (Rondani) (: ) stands out among the factors limiting Brazilian wheat production. Chemical control is predominant in management making the production dependent on insecticides. The effect of silicon and previous infestation with aphids on the induction of resistance to the greenbug was evaluated in wheat plants. Treatments consisted of control; fertilization with calcium silicate; plant infestation with aphids; fertilization with calcium silicate + plant infestation with aphids. A free-choice preference test was performed 35 days after seedling emergence; the

aphid’s intrinsic rate of population increase (rm) was also determined, and the activities of three enzymes (peroxidase, polyphenoloxidase, and phenylalanine ammonia-lyase) involved in plant defense were quantified. Silicon fertilization and the previous infestation with aphids induced wheat plant resistance to the greenbug. Key words: , Triticum aestivum, pest management, induced resistance, host plant resistance

INDUÇÃO DE RESISTÊNCIA EM PLANTAS DE TRIGO POR SILÍCIO E PULGÕES

RESUMO: Dentre os fatores limitantes da produção tritícola brasileira está o ataque de insetos-praga, podendo- se destacar o pulgão-das-gramíneas Schizaphis graminum (Rondani) (Hemiptera: Aphididae). O controle químico é predominante no manejo desse pulgão, tornando a produção dependente do uso de inseticidas. Este trabalho avalia o efeito do silício e da infestação prévia com pulgões na indução de resistência ao pulgão-das- gramíneas em plantas de trigo. Os tratamentos foram: testemunha; adubação com silicato de cálcio; infestação das plantas com pulgões; adubação com silicato de cálcio + infestação das plantas com pulgões. Trinta e cinco dias após emergência das plântulas foi realizado teste de preferência com chance de escolha; determinação

da taxa de crescimento da população do pulgão (rm) e quantificação da atividade de três enzimas envolvidas na defesa das plantas: peroxidase, polifenoloxidase e fenilalanina amônia-liase. A adubação silicatada e a infestação prévia com pulgões induzem resistência em plantas de trigo ao pulgão-das-gramíneas. Palavras-chave: Schizaphis graminum, Triticum aestivum, manejo de pragas, resistência induzida, resistência de plantas a insetos

INTRODUCTION Fawe et al., 1998). However, silicon has not yet been in- dicated as a plant defense response elicitor against the in- Silicon is not considered an essential element for sect attack. most plants, but it has been reported that absorption of Elicitors trigger the induced resistance process; silicon is beneficial to crops, hence induces protection they are agents that induce any defense response in the against pest attack (Epstein, 1994; 1999). The protection plants (Dixon et al., 1994). Increased activity of defen- conferred to plants by silicon could be related to its ac- sives enzymes such as peroxidase (POX), cumulation and polymerization in the cells, forming a polyphenoloxidase (PPO), and phenylalanine ammonia- mechanical barrier that difficult the -pests attack lyase (PAL), are events related to resistance inducement (Yoshida et al., 1962). However, mechanical barriers are in plants (Karban & Myers, 1989). The peroxidases are not the only defense mechanism against external agents. involved in the processes of lignification (Goodman et Studies with cucumber (Cucumis sativus L.) have shown al., 1986) and suberization (Bowles, 1990). The enhanced resistance of silicon-treated plants to Pythium polyphenoloxidase enzyme catalizes the oxidation of phe- spp. and Sphaerotheca fuliginea (Schlecht.) Poll. result- nolic compounds to quinones, thus decreasing the nutri- ing from the accumulation of phenolic compounds, lig- tional quality of food and reducing its protein digestibil- nin and phytoalexins (Chérif et al., 1992a; 1992b; 1994; ity (Felton & Duffey, 1990; Felton et al., 1994). The in-

Sci. Agric. (Piracicaba, Braz.), v.62, n.6, p.547-551, Nov./Dec. 2005 548 Gomes et al. tensification of production of phenolic compounds, Greenbug’s rate of population increase - Twenty four known as defense molecules of plants against pathogens hour-old nymphs were individualized in clear, plastic cy- and , is indicated by an increase in phenylalanine lindric cages (1 cm height; 0.8 cm in diameter), with the ammonia-lyase activity in wounded plant tissues (Bi & bottom closed with organdy fabric and the rim on the Felton, 1995). Therefore, the objective of this work was other end padded with foam. Three cages were installed to evaluate the effect of silicon and of previous infesta- per plant and per pot, and the mean value was later cal- tion with aphids on the induction of resistance to the culated. The method proposed by Wyatt & White (1977) greenbug, Schizaphis graminum (Rondani) (Hemiptera: was utilized to estimate the insect’s population increase Aphididae), in wheat plants. rate.

MATERIAL AND METHODS Enzymatic activity - Wheat leaves were macerated with a mortar and pestle in the presence of liquid nitrogen, and -1 Plants and insects - Wheat plants (Triticum aestivum 10 mL of phosphate-potassium buffer (0.1 mol L ; pH L. cv. Embrapa 42) were grown in greenhouse condi- 6.1) were added to 0.2 g of macerated leaves. After rest- o tions in 4-kg soil capacity pots for 90 days. All fertili- ing for one hour at 4 C and periodical agitation, the so- o zation procedures were performed, based on soil analy- lution was centrifuged at 13,000 g for 15 minutes, at 4 C. sis (Malavolta, 1980). Each pot was covered by an or- Supernatants (enzyme extracts) were then utilized for en- gandy fabric cage (40 cm width ´ 40 cm length ´ 80 zymatic activity determination. Peroxidade (POX) and cm height). Moisture in the pots was provided and main- polyphenoloxidase (PPO) activities were measured by tained with daily irrigations. Plants fertilized with cal- spectrophotometry through the increase in optical density -1 -1 -1 -1 -1 cium silicate received 1.855 g kg soil. The calcium sili- (OD), using OD470 min g and OD420 min g , respec- tively, following methodology adapted from Silva (2002). cate contained 62% SiO2 and 18% CaO. Plants which did not receive calcium silicate were fertilized with cal- The substrates utilized by POX and PPO were guaiacol/ -1 cium carbonate, 0.61g kg soil. The aphids S. graminum H2O2, and catechol, respectively. The readings were per- were taken from a mass rearing, maintained on variety formed each second, for 2 minutes and the activities were -1 Lorini wheat plants. expressed as units per gram of fresh weight (u g ). One activity unit was defined as the increment of 0.1 absor- Treatments - Treatments consisted of: 1- zero calcium bance unit per minute. To determine the phenylalanine silicate and absence of aphids (control); 2- fertilization ammonia-lyase (PAL) activity, the enzyme extract was with calcium silicate and absence of aphids; 3- infesta- prepared with 1 g of macerated leaves, 5 mL tris-HCL tion of plants at 30 days after emergence 30 adult aphids 0.1 mol L-1 buffer, pH 8.8, and 0.1 g insoluble polyvi- per plant for five days, and no calcium silicate fertiliza- nylpyrrolidone. The buffer contained 10-3mol L-1 EDTA tion; and, 4- fertilization with calcium silicate plus infes- and 10-2 mol L-1 b-mercaptoethanol. After resting for one tation of plants at 30 days after emergence, 30 adult hour at 4ºC and periodical agitation, the solution was aphids per plant for five days. centrifuged at 1,500 g for 5 minutes, at 4oC. The super- natants (enzyme extracts) were utilized for enzymatic Evaluations were made 35 days after seedling activity determination. PAL activity was measured by emergency included: -1 spectrophotometry through the increase in OD280 min Preference test with leaves removed from wheat g-1, following methodology adapted from Data & plants - Petri dishes (20 cm) were utilized as arenas. Quevedo (1984). The vials containing the reaction mix- Dishes had bottoms lined with filter paper moistened ture were incubated in water bath at 37oC for five peri- with distilled water; each dish received one, 8-cm long ods of time (0, 30, 60, 90, and 120 minutes), with 0.5 -1 leaf cut of each treatment, immersed in a 10-6 mol L-1 mL perchloric acid 2 mol L utilized to halt the reac- benzyladenine solution, inside a 3 cm long, 0.5 cm di- tion. Activity was expressed as activity units per gram -1 ameter plastic straw, plugged with cotton and randomly of fresh weight (u g ). One unit was defined as the arranged side by side. The leaf cut was obtained from amount of trans-cinnamic acid formed per minute un- the plant’s first completely unfolded leaf. Twenty adult der the assay conditions. aphids were released at straw’s opposite side. Dishes Statistics - Four treatments and ten replicates were uti- were them sealed with perforated plastic film and placed o lized. Data obtained were submitted to ANOVA, and the over a lab bench, in an air-conditioned room (26 ± 2 C), means compared by Scott & Knott test (1974) (P = 0.05). relative humidity 70 ± 10%, and 12-hour photophase. Evaluations were performed 24, 48, and 72 hours after RESULTS releasing the aphids by counting the number of adult aphids and nymphs (removed after counting) on each A reduction in total number of aphids was ob- leaf cut. served at 48 and 72 hours for treatments in which plants

Sci. Agric. (Piracicaba, Braz.), v.62, n.6, p.547-551, Nov./Dec. 2005 Resistance induction in wheat plants 549 were fertilized with silicon. At 72 hours, leaf cuts of The greatest POX activity occurred in the treat- plants that received silicon fertilization, or silicon com- ment with silicon fertilization and pre-infestation (602 u bined with a pre-infestation, were less colonized by g-1 fresh weight), followed by the treatments with pre-in- aphids (Table 1). festation, and silicon fertilization (441 and 268 u g-1 fresh The lowest rate of greenbug population increase weight, respectively). All treatments showed higher ac- -1 (rm = 0.14) occurred in the treatment with aphid pre-in- tivities than the control (137 u g fresh weight) (Table festation and silicon fertilization, while the highest popu- 2). Treatment silicon fertilization plus pre-infestation (489 -1 lation increase was observed in the control (rm = 0.37). u g fresh weight) showed higher PPO activity than the The rate of S. graminum population increase in the treat- treatments with silicon fertilization and pre-infestation, ments with silicon or pre-infestation was intermediate which had an intermediate responce (251 and 272 u g-1 between these two extremes (rm = 0.25 and rm = 0.23, re- fresh weight, respectively), although they were still higher spectively) (Figure 1). than the control (135 u g-1 fresh weight) (Table 2). Only the pre-infestation treatment and the treatment in which 0.4 silicon fertilization was combined with pre-infestation -1

) Control (0.049 and 0.127 u g fresh weight, respectively) led to m

r Silicon fertilization (

an increase in PAL activity, while the treatment with sili-

e 0.3 Pre-infestation with aphids s con alone did not differ from the control (Table 2). a

e Silicon + aphids r c n i

DISCUSSION n o

i 0.2 t a l Pre-infestation with aphids and/or silicon appli- u p

o cation induce defenses in wheat plants that will affect p

f

o preference and population increase in S. graminum, es- 0.1 e t pecially when they act jointly, with cummulative effects. a

R Silicon-fertilized plants present changes in their trichomes (Samuels et al., 1993), as well as in their epidermis and 0 Treatments internal tissues (Hodson & Sangster, 1988). Research on the distribution of silicon indicated that it can be depos- Figure 1 - Rate of population increase (rm) of the greenbug, Schizaphis graminum (mean ± standard error) in the ited, in the form of silica, in the intracellular spaces, in- greenhouse. Bars sharing same letters do not differ by side the cells, and in the cell wall (Kaufman et al., 1985), Scott & Knott test (P < 0.05). as well as in conducting vessels (Hayward & Parry, 1973).

Table 1 - Total number (mean ± standard error) of Schizaphis graminum aphids on leaves removed wheat plants. Total number of aphids per leaf Treatment 24 hours 48 hours 72 hours Control 24.6 ± 4.04 aA 35.7 ± 4.55 aA 30.6 ± 1.96 aA Silicon fertilization (SF) 16.2 ± 2.95 aA 11.6 ± 1.75 bB 7.3 ± 1.21 cB Pre-infestation with aphids (PIA) 15.7 ± 2.11 aA 15.9 ± 2.73 bA 13.6 ± 1.71 bA SF + PIA 17.8 ± 3.58 aA 5.0 ± 1.91 cB 5.2 ± 1.84 cB Means followed by a common, lower case letter in the column, and by a common, upper case letter in the row, do not differ by Scott & Knott test (P < 0.05).

Table 2 - Peroxidase (POX), polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities (mean ± standard error) in wheat plants. Enzymatic activity Treatment POX PPO PAL ------u g-1 fresh weight ------Control 137 ± 15 d 135 ± 23 c 0.017 ± 0.002 c Silicon fertilization (SF) 268 ± 25 c 251 ± 20 b 0.018 ± 0.001 c Pre-infestation with aphids (PIA) 441 ± 46 b 272 ± 43 b 0.049 ± 0.002 b SF + PIA 602 ± 68 a 489 ± 29 a 0.127 ± 0.002 a Means followed by a common lower case letter in the column do not differ by Scott & Knott test (P < 0.05).

Sci. Agric. (Piracicaba, Braz.), v.62, n.6, p.547-551, Nov./Dec. 2005 550 Gomes et al.

Results obtained herein might be related to the deposi- of these enzymes indicates the synthesis of plant defense tion of silica in the leaf tissue which, by increasing leaf compounds against external agents. hardness, makes it difficult for the insect’s stylet to pen- etrate the leaf. However, other researches have demon- ACKNOWLEDGEMENTS strated that the application of silicon could change the physiological properties of plants. To Universidade Federal de Lavras and Fundação Chérif et al. (1994) observed that cucumber plants de Amparo à Pesquisa do Estado de Minas Gerais fertilized with silicon showed an increase in the synthe- (FAPEMIG) for making this research work possible, and sis of the enzymes peroxidase, polyphenoloxidase, b-1,3 to Conselho Nacional de Desenvolvimento Científico e glucanase, and chitinase, as a response to infection by Tecnológico (CNPq) for granting a scholarship. pathogens. 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