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The Content of Bound Amino Acids in Lucerne Plants Infected with the Bacterial Wilt
Zbl. Bakt. II. Abt. 136 (1981), 359-365 [From the Research Institute of Plant Production, Praha-Ruzhne]
The Content of Bound Amino Acids in Lucerne Plants Infected with the Bacterial Wilt V.
KUDELA
and J.
KRATKA
With 3 Figures
Summary Lucerne (Medicago Bativa L.) seedlings of relatively resistant cultivar Hodonfnska (R-cultivar) and those of very susceptible cultivar Pferovska (S-cultivar) were inoculated at the stage of cotyledons with Oornynebacterium inBidioBUm (McCulloch) Jensen. The plants were analysed on the 20th, 35th, and 50th day after inoculation. After the acid hydrolysis with 6 N HCl, the content of bound amino acids was determined using an automatic analyser. The total amino acid content, especially of proline, glycine, alanine, phenylalanine, and isoleucine, and in the case of the R-cultivar also the histidine content was increased, due to the infection. The increase in total amino acid content, due to the infection and the change in the content of individual amino acids, were in R-cultivar most pronounced on the 20th day and in S-cultivar plants on the 35th day after inoculatin. The amino acid content of inoculated plants dropped to the level of control plants on the 50th day after inoculation.
Zusammenfassung Die verhaltnismaLlig widerstandefahige Luzernesorte Hodonfnsks (R-Sorte) und die auLlerst anfalligs Sorte Prerovske. (S-Sorte) wurden im Keimblattstadium mit Corynebacterium inBidioBUm (McCulloch) Jensen infiziert. Am 20., 35. und 50. Tag nach der Beimpfung wurden die Pflanzen analysiert. Nach Hydrolyse des Materials mit 6 N HCl bei 120°C innerhalb von 24 Std. wurde der Aminosauregehalt im Aminosaureanalysator ermittelt. Durch die Infektion erhohte sich der Gesamtgehalt an Aminoaauren, besonders aber der an Prolin, Glyzin, Alanin, Glutaminsaure, Phenylalanin und Isoleuzin; bei der R-Sorte auch der Gehalt an Histidin. Der Aminosaureanstieg und die wesentlichsten Veranderungen im Gehalt an einzelnen Aminosauren, durch die Infektion hervorgerufen, waren bei der R-Sorte am 20. Tag und bei der S-Sorte am 35 Tag am deutlichsten ausgepregt, Am 50. Tag nach der Inokulation ging der Aminosauregehalt auf das Niveau der Kontrollpflanzen zuruck,
Recently considerable attention has been paid both to the biocidic and/or inhibitory effects of amino acids on the plant pathogens and to the possibilities of influencing the amino acid metabolism of the host plant to obtain resistance effects. Partial succsess (e.g., with apple scab) raised the hope on the use of amino acids in chemotherapy. In some cases, correlation between plant resistance, and the content of single or several amino acids could be found (VAN ANDEL 1966). Several authors (e.g. HEK· KERT et al. 1975) attempted to classify the resistance and susceptibility in non-infected plants according to the content of individual amino acids and their relations.
360
V. KUDEI,A and J. KRATKA
The role of proteins has been emphasized recently in investigat ions of mutual hostparasite relationships because of t heir importance in immunological processes . Specificity of the interrelationships between the host and the parasite was explained by the existence of common antigen (DE YAY et al. 1972). Investigating the lucerne bacterial wilt pathogenesis, we determined the changes in the content of bound amino acids caused by infection. Materials and Methods Lucerne (.ltledicago sat i va L.) plants wer e grown in shallow vegetation pots filled with sili ca sand, su ppleme nted with Hellriegel's nutrien t solution. The oult ivar Psflo vska whi ch is very susceptible to the bacterial wilt and the eultdvar Hodonfnska which is relatively resistant to this pathogen (K UDELA 1971) ser ved as experiment al plants. They wer e inoculated with Ooryn ebacterium insidi o8um (McCulloch) J ensen at the stage of cotyledons, using the method described by KREITLOW (1963). Both inoculated and cont r ol plants were sampled for analyses on the 20th, 35th, an d 50th day after ino eult.ion. At each sampling inoculated plants as well as control plants were an alysed in the amount of 50 to 60. Plant dry weight (expressed per on e plant) was determined in eac h experimental variant. Plant sampling dates were chosen in su ch a way that the first sampling covered the stage preceding the appearance of the first visible symptoms of the dis ease in the leafs of susceptible pl ants an d the third sampling coincided with the period of full symptom m anifestation. In the course of the experiment, i.e. from the end of September till mid-December, the experimental plants were grown in a gl asshouse with additional artificial light (8,000 lx, 12 h), tempera· ture being between 21 and 25 °C during the day an d 15 to 18 °C at night. Amino acid content was determined by me ans of an automatic anal ys er (CSA V, type 6020). Bound a m ino acid con ten t was determined after the sample hydrolysi s with 6 N HCI for 24 h at 120 °C and the result obtained were expressed in mg . g-l of the dry m atter. Differ en ces in the amino acid content between the both cultivars wer e determined by calculating the r atio Hodoninsk a: Prero vaka.
Results
Comparisons of t he dry weight of the control (healthy) and the inoculated (infected) plants (Fig. 1) make possible to show the influence of infection on the total amount of synthesized proteins and to express quantitatively the differences in the degree of resistance between the two investigated cultivars. The difference in dry weight of the infected plants between the two oultivars increased with the time after inoculation, becaus e the retardation of growth of the infected Pferovska plants was mor e pronounced than that of infected Hodoninska plants. Infected plants of both the cultivars showed higher content of total bound amino acids (expressed per unit of dry matter) than the control plants at all three sampling dates. This increase was most conspicuous an the 20th day after inoculation in infected Hodoninska plants, while in Pferovska plants it occurred later, on the 35th day. The total amino acid content of infected plants of both cultivars resembled that of control plants on the 50th day after inoculation (Fig. 1). Changes in the content of individual amino acids The changes in the content of individual amino acids in infected and control plants of both the cultivars are presented in Fig. 2. The changes caused by the infection were either consistent in the both cultivars (e.g., increase in proline and methionine contents on the 20th day after inoculation), or they were divergent. For instance in cv. Hodoninska the increase in arginine content and in cv. Prerovska the decrease in histidine, serin e, threonine, and valine content were pronounced on the 20th day after inoculation.
The Content of Bound Amino Acids in Lucerne Plants
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Fig. 1. Comparison of dry weight (left) un d of total amino acid content (right) between infected (inoculated) and healthy (not inoeul ated) pl unts on the 20th, 35th, an d 50th day after inoculation.
The increase in proline, glycine, alanine, phenylalanine, and isoleucine content" due to the infection, was most pronounced in both cultivars at all three sampling dates. The higher histidine content of infected Hodoninska plants in comparison to the control plants could be noticed at all sa mpling dates. In infected Hodoninska plants the tyrosine and serine dropped below the level of the control plants on the 35th day after inoculation, the serine content dropped on the 50th da y as well. Difference s between Cultivars Control Hodoninska plants had a higher total amino acid content than control Pferovska plants at the first and second sampling date (at the age of 27 or 42 days, respectively). At the third sampling date (at the age of 57 days), the differences between the two cultivars were negligible (Fig. 3). Control Hodoninska plants showed higher methionine and proline content than control Pferovska plants at all sampling dates and higher content, particularly of threonine, aspartic acid, phenylalanine, and serine at the first sampling date (Fig. 2). Infected Hodoninska plants contained by 30 % more amino acids than the infect ed Pferovsks plants an the 20th day. After 35 days the amino acid content in Pferovska plants increased due to infection, while it dropped in Hodoninska plants. Thus the cv. Pferovska showed by 8 % higher amino acid content than the cv. Hodoninska. After 50 days the total amino acid content was almost equal in the infected plants of the both cultivars (Fig. 3). On the 20th day the arginine, threonine, valine, serine, and proline content were found by 50 % higher and that of tyrosine, phenylalanine, glutamic acid, alanine, and aspartic acid by 25 % higher in the infected Hodoninska than in Prerovska plants. Changes in the content of individual amino acids in Prerovska plants, caused by the infection during 35 days after inoculation, resembled those found in Hodoninska plants caused during 20 days after inoculation; especially proline, glycine, threonine, phenylalanine, alanine, glutamic acid, leucine, and serine reached high values as com-
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The Content of Bound Amino Acids in Lucerne Plants
363
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Fig. 3. Comparison of the total conten t 'of boun d amin o acids in cv, H odonfnsk a and cv, Pferovska on the 50th d ay after inoculat ion.
pared with t he control. At t he 35th day after inoculation, t he content of most ami no acids was higher in infected P rerovska t han in Hodonfnska plants, t he differences in t he serine, t hreonine, glutamic acid, ph enylalanine, proline, tyrosine, and alanine contents were most appa rent. Higher histidine and lower serine and aspartic acid content in infected Hodoninska plants, as compa red with infected Pferovska plan ts, was remarkabl e at t he 50th day after inoculat ion (Fig. 2). Discussion The increase in bound am ino acid content per gram of dr y matter, which was found in lucerne plants infect ed with t he bact erial wilt, corresponds with the results obtained by oth er authors investigating other t rac heobacterioses and tracheomycoses, e.g, studies by SATYANARYAN (1955) (after SADAVISAN 1965) in cotton plant s infected with F usarium vasintectum, PEGG and SEQUEIRA (1968) studies in t obacco plant s infected with P seudomonas solanacearum, and RAGGI et al. (1974) in tomato plant s infect ed with F usarium oxysporum f. lqcopereici. In progressive stages of t he infection suscept ible plants usually showed a decrease in protein content. Such decrease was never observed in our experiment s up to t he 50th day after inoculation. Th is can be explained by a relatively slow course of bact erial wilt disease in lucerne plants. The symptoms or even t he factors related to the plant resistance were determined in our experiment as follows : (1) high total amino acid content, especially proline and perh aps methionine ; (2) enhanced prot ein synthesis and a shift in pro tein pat tern (judged according to changes in t he ratio of individual amino acids) in t he early phases of growth after infecti on, i.e, before t ho appearance of t he visible sym ptoms of t he disease in susceptible plants ; (3) decreased tyrosine and serine content between t he 20th and 30th da y after inoculati on.
Fig. 2. Conten t of bound amino a cids in infected (inocul ated) and he althy (not inocul ated) plan t s cv, Hodonfnsk a (H) and Pfer ovsk a (P).
364
Y. KUDELA and J. KRATKA
The response to unfection of the very susceptible cultivar Pferovska was characterized as follows: (1) decreased histidine, serine, threonine, and valine content below the level in control plants and slightly increased total amino acid content after infection (before the visible symptoms in the leaves appear); (2) enhanced protein accumulation at later stages after inoculation as compared with relative resistant Hodoninska plants. According to RUBIN (1975) the enhanced protein synthesis is viewed as an active response of host plants to the invasion of either obligate or facultative parasites. The ribosomes of the infected resistant plants tend to increase and, hence, the efficiency of the protein synthesis is higher in the infected than in the non-infected plants. RAGGl et al. (1974) reported that the initial increase on protein content in infected tissues was connected with an increased content of the growth-promoting substances (auxin, kinetin) which may enhance the amino acid incorporation into proteins. After an initial increase, the serine and tyrosine content decreased in infected plants of relatively resistant cultivar Hodoninska below the level of the control. This might be in connection with their utilization of serine for the synthesis of tryptophane and auxin, the concentration of which in resistant plants sharply increases an the 14th day after inoculation (KRATKA and KUDELA, in press). The decrease in tyrosine content might be connected with role in the synthesis of phenolic compounds, melanines, etc., which are known to be involved in plant-defensive reactions (KOSUGE 1969). The greatest changes both in the quantity of synthesized proteins and in the protein pattern, as caused by lucerne bacterial wilt, were recorded in plants of the relatively resistant cultivar on the 20th day and in plants of the susceptible cultivar on the 35th day after inoculation. The experimental results support the hypothesis that the resistance or susceptibility degrees are determined by the rate of plant response to the presence of the pathogen; an immediate activation of alternative metabolic pathways causes resistance, the retarded activation causes susceptibility. This hypothesis is also supported by protein changes, observed in lucerne plants infected with Verticillium albo-atrum (WHITHEY, HEALE and VAUGHAN 1972). Analysis of the infected materials by means of the disc electrophoresis gave evidence of the progressive alternationsin the protein composition in the stems of the susceptible cultivars, whereas the strong initial reaction of resistant cultivar, manifested by the change of the protein structure, turned back towards normal development.
References DE YAY, J. E., CHARUDATTAN, R., and WIMALAJEEWA, D. L. S.: Common antigenic determinants as a possible regulator of host-pathogen compatibility. Amer. Naturalist 106 (1972), 185-194. HECKER, R. J., RUPPEL, E. G., MAAG, G. W., and RASMUSON, D. M.: Amino acids associated with Cercospora leaf spot resistance in sugar beet. Phytopath. Z. 82 (1975),175-181. HEITEFUSS, R.: Fortschritte in der Analyse der Wirt-Parasit-Beziehungen. Angew. Botanik 53 (1979),111-125. KOSUGE, T.: The role of phenolics in host response to infection. Ann. Rev. Phytopath. 7 (1969), 195-222. KREITLOW, K. W.: Infecting of seven-day-old alfalfa seedlings with wilt bacteria through wounded cotyledons. Phytopathology 53 (1963), 800 - 803. KUDELA, V.: Odolnost eeskoslovenskvch odrud vojtesky proti bakterialnfmu vadnutf (Resistance of Czechoslovak lucerne varieties to bacterial wilt). Ochr, Rostl. 7 (XLIV) (1971),169-177. PEGG, G. F., and SEQUEIRA, L.: Stimulation of aeromatic biosynthesis in tobaco plants infected by Pseudomonas solanaceorum: Phytopathology 58 (1968),476-483. RAGGI, V., ZAZZERINI, A., BARBERINI, B., FERBANTI, F., and DBAOLI, R.: Alterations in the composition of free and combined amino acids in susceptible and resistant tomato cultivars infected with Fusarium oyysporum f. lycopersici. Phytopath. Z. 79 (1974), 258 -280.
The Content of Bound Amino Acids in Lucerne Plants
365
RUBIN, B. A.: Problemy biochimii patogeneza i immuniteta rastenij (Biochemical problems of pathogenesis and immunity). In: Immunitet selskoehosjajstvennyeh rastenij k boleznjam i vrediteljam. Moskva (1975), 158-166. SADASIVAN, F. S.: Physiology of wilting plants. In: Biochemische Problerne del' kranken Pflanze. Tagungsbericht Nr. 74. Dtsch. Akad. Landwirtschaftswiss. Berlin (1965), 149 -163. ANDEL, O. M. VAN: Amino acids and plant diseases. Ann. Rev. Phytopathol. 4 (1966), 349-368. ·WHITNEY, P.J., REALE, J. B., and VAUGHAN, J. G.: Protein changes in the vascular wilt disease of lucerne caused by Verticillium albo-atrum R. et B. J. expel'. Bot. 23 (1972), 400-414. Authors' address: Ing. VACLAV KUDELA, CSc., and RNDr. JrRINA KBATKA, CSc., Research Institute for Plant Product.ion CS -161 06 Prague 6·Ruzyne 507, Czechoslovakia.
The Content of Bound Amino Acids in Lucerne Plants Infected with the Bacterial Wilt V.
KUDELA
and J.
KRATKA
With 3 Figures
Summary Lucerne (Medicago Bativa L.) seedlings of relatively resistant cultivar Hodonfnska (R-cultivar) and those of very susceptible cultivar Pferovska (S-cultivar) were inoculated at the stage of cotyledons with Oornynebacterium inBidioBUm (McCulloch) Jensen. The plants were analysed on the 20th, 35th, and 50th day after inoculation. After the acid hydrolysis with 6 N HCl, the content of bound amino acids was determined using an automatic analyser. The total amino acid content, especially of proline, glycine, alanine, phenylalanine, and isoleucine, and in the case of the R-cultivar also the histidine content was increased, due to the infection. The increase in total amino acid content, due to the infection and the change in the content of individual amino acids, were in R-cultivar most pronounced on the 20th day and in S-cultivar plants on the 35th day after inoculatin. The amino acid content of inoculated plants dropped to the level of control plants on the 50th day after inoculation.
Zusammenfassung Die verhaltnismaLlig widerstandefahige Luzernesorte Hodonfnsks (R-Sorte) und die auLlerst anfalligs Sorte Prerovske. (S-Sorte) wurden im Keimblattstadium mit Corynebacterium inBidioBUm (McCulloch) Jensen infiziert. Am 20., 35. und 50. Tag nach der Beimpfung wurden die Pflanzen analysiert. Nach Hydrolyse des Materials mit 6 N HCl bei 120°C innerhalb von 24 Std. wurde der Aminosauregehalt im Aminosaureanalysator ermittelt. Durch die Infektion erhohte sich der Gesamtgehalt an Aminoaauren, besonders aber der an Prolin, Glyzin, Alanin, Glutaminsaure, Phenylalanin und Isoleuzin; bei der R-Sorte auch der Gehalt an Histidin. Der Aminosaureanstieg und die wesentlichsten Veranderungen im Gehalt an einzelnen Aminosauren, durch die Infektion hervorgerufen, waren bei der R-Sorte am 20. Tag und bei der S-Sorte am 35 Tag am deutlichsten ausgepregt, Am 50. Tag nach der Inokulation ging der Aminosauregehalt auf das Niveau der Kontrollpflanzen zuruck,
Recently considerable attention has been paid both to the biocidic and/or inhibitory effects of amino acids on the plant pathogens and to the possibilities of influencing the amino acid metabolism of the host plant to obtain resistance effects. Partial succsess (e.g., with apple scab) raised the hope on the use of amino acids in chemotherapy. In some cases, correlation between plant resistance, and the content of single or several amino acids could be found (VAN ANDEL 1966). Several authors (e.g. HEK· KERT et al. 1975) attempted to classify the resistance and susceptibility in non-infected plants according to the content of individual amino acids and their relations.
360
V. KUDEI,A and J. KRATKA
The role of proteins has been emphasized recently in investigat ions of mutual hostparasite relationships because of t heir importance in immunological processes . Specificity of the interrelationships between the host and the parasite was explained by the existence of common antigen (DE YAY et al. 1972). Investigating the lucerne bacterial wilt pathogenesis, we determined the changes in the content of bound amino acids caused by infection. Materials and Methods Lucerne (.ltledicago sat i va L.) plants wer e grown in shallow vegetation pots filled with sili ca sand, su ppleme nted with Hellriegel's nutrien t solution. The oult ivar Psflo vska whi ch is very susceptible to the bacterial wilt and the eultdvar Hodonfnska which is relatively resistant to this pathogen (K UDELA 1971) ser ved as experiment al plants. They wer e inoculated with Ooryn ebacterium insidi o8um (McCulloch) J ensen at the stage of cotyledons, using the method described by KREITLOW (1963). Both inoculated and cont r ol plants were sampled for analyses on the 20th, 35th, an d 50th day after ino eult.ion. At each sampling inoculated plants as well as control plants were an alysed in the amount of 50 to 60. Plant dry weight (expressed per on e plant) was determined in eac h experimental variant. Plant sampling dates were chosen in su ch a way that the first sampling covered the stage preceding the appearance of the first visible symptoms of the dis ease in the leafs of susceptible pl ants an d the third sampling coincided with the period of full symptom m anifestation. In the course of the experiment, i.e. from the end of September till mid-December, the experimental plants were grown in a gl asshouse with additional artificial light (8,000 lx, 12 h), tempera· ture being between 21 and 25 °C during the day an d 15 to 18 °C at night. Amino acid content was determined by me ans of an automatic anal ys er (CSA V, type 6020). Bound a m ino acid con ten t was determined after the sample hydrolysi s with 6 N HCI for 24 h at 120 °C and the result obtained were expressed in mg . g-l of the dry m atter. Differ en ces in the amino acid content between the both cultivars wer e determined by calculating the r atio Hodoninsk a: Prero vaka.
Results
Comparisons of t he dry weight of the control (healthy) and the inoculated (infected) plants (Fig. 1) make possible to show the influence of infection on the total amount of synthesized proteins and to express quantitatively the differences in the degree of resistance between the two investigated cultivars. The difference in dry weight of the infected plants between the two oultivars increased with the time after inoculation, becaus e the retardation of growth of the infected Pferovska plants was mor e pronounced than that of infected Hodoninska plants. Infected plants of both the cultivars showed higher content of total bound amino acids (expressed per unit of dry matter) than the control plants at all three sampling dates. This increase was most conspicuous an the 20th day after inoculation in infected Hodoninska plants, while in Pferovska plants it occurred later, on the 35th day. The total amino acid content of infected plants of both cultivars resembled that of control plants on the 50th day after inoculation (Fig. 1). Changes in the content of individual amino acids The changes in the content of individual amino acids in infected and control plants of both the cultivars are presented in Fig. 2. The changes caused by the infection were either consistent in the both cultivars (e.g., increase in proline and methionine contents on the 20th day after inoculation), or they were divergent. For instance in cv. Hodoninska the increase in arginine content and in cv. Prerovska the decrease in histidine, serin e, threonine, and valine content were pronounced on the 20th day after inoculation.
The Content of Bound Amino Acids in Lucerne Plants
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100
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Fig. 1. Comparison of dry weight (left) un d of total amino acid content (right) between infected (inoculated) and healthy (not inoeul ated) pl unts on the 20th, 35th, an d 50th day after inoculation.
The increase in proline, glycine, alanine, phenylalanine, and isoleucine content" due to the infection, was most pronounced in both cultivars at all three sampling dates. The higher histidine content of infected Hodoninska plants in comparison to the control plants could be noticed at all sa mpling dates. In infected Hodoninska plants the tyrosine and serine dropped below the level of the control plants on the 35th day after inoculation, the serine content dropped on the 50th da y as well. Difference s between Cultivars Control Hodoninska plants had a higher total amino acid content than control Pferovska plants at the first and second sampling date (at the age of 27 or 42 days, respectively). At the third sampling date (at the age of 57 days), the differences between the two cultivars were negligible (Fig. 3). Control Hodoninska plants showed higher methionine and proline content than control Pferovska plants at all sampling dates and higher content, particularly of threonine, aspartic acid, phenylalanine, and serine at the first sampling date (Fig. 2). Infected Hodoninska plants contained by 30 % more amino acids than the infect ed Pferovsks plants an the 20th day. After 35 days the amino acid content in Pferovska plants increased due to infection, while it dropped in Hodoninska plants. Thus the cv. Pferovska showed by 8 % higher amino acid content than the cv. Hodoninska. After 50 days the total amino acid content was almost equal in the infected plants of the both cultivars (Fig. 3). On the 20th day the arginine, threonine, valine, serine, and proline content were found by 50 % higher and that of tyrosine, phenylalanine, glutamic acid, alanine, and aspartic acid by 25 % higher in the infected Hodoninska than in Prerovska plants. Changes in the content of individual amino acids in Prerovska plants, caused by the infection during 35 days after inoculation, resembled those found in Hodoninska plants caused during 20 days after inoculation; especially proline, glycine, threonine, phenylalanine, alanine, glutamic acid, leucine, and serine reached high values as com-
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The Content of Bound Amino Acids in Lucerne Plants
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Fig. 3. Comparison of the total conten t 'of boun d amin o acids in cv, H odonfnsk a and cv, Pferovska on the 50th d ay after inoculat ion.
pared with t he control. At t he 35th day after inoculation, t he content of most ami no acids was higher in infected P rerovska t han in Hodonfnska plants, t he differences in t he serine, t hreonine, glutamic acid, ph enylalanine, proline, tyrosine, and alanine contents were most appa rent. Higher histidine and lower serine and aspartic acid content in infected Hodoninska plants, as compa red with infected Pferovska plan ts, was remarkabl e at t he 50th day after inoculat ion (Fig. 2). Discussion The increase in bound am ino acid content per gram of dr y matter, which was found in lucerne plants infect ed with t he bact erial wilt, corresponds with the results obtained by oth er authors investigating other t rac heobacterioses and tracheomycoses, e.g, studies by SATYANARYAN (1955) (after SADAVISAN 1965) in cotton plant s infected with F usarium vasintectum, PEGG and SEQUEIRA (1968) studies in t obacco plant s infected with P seudomonas solanacearum, and RAGGI et al. (1974) in tomato plant s infect ed with F usarium oxysporum f. lqcopereici. In progressive stages of t he infection suscept ible plants usually showed a decrease in protein content. Such decrease was never observed in our experiment s up to t he 50th day after inoculation. Th is can be explained by a relatively slow course of bact erial wilt disease in lucerne plants. The symptoms or even t he factors related to the plant resistance were determined in our experiment as follows : (1) high total amino acid content, especially proline and perh aps methionine ; (2) enhanced prot ein synthesis and a shift in pro tein pat tern (judged according to changes in t he ratio of individual amino acids) in t he early phases of growth after infecti on, i.e, before t ho appearance of t he visible sym ptoms of t he disease in susceptible plants ; (3) decreased tyrosine and serine content between t he 20th and 30th da y after inoculati on.
Fig. 2. Conten t of bound amino a cids in infected (inocul ated) and he althy (not inocul ated) plan t s cv, Hodonfnsk a (H) and Pfer ovsk a (P).
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The response to unfection of the very susceptible cultivar Pferovska was characterized as follows: (1) decreased histidine, serine, threonine, and valine content below the level in control plants and slightly increased total amino acid content after infection (before the visible symptoms in the leaves appear); (2) enhanced protein accumulation at later stages after inoculation as compared with relative resistant Hodoninska plants. According to RUBIN (1975) the enhanced protein synthesis is viewed as an active response of host plants to the invasion of either obligate or facultative parasites. The ribosomes of the infected resistant plants tend to increase and, hence, the efficiency of the protein synthesis is higher in the infected than in the non-infected plants. RAGGl et al. (1974) reported that the initial increase on protein content in infected tissues was connected with an increased content of the growth-promoting substances (auxin, kinetin) which may enhance the amino acid incorporation into proteins. After an initial increase, the serine and tyrosine content decreased in infected plants of relatively resistant cultivar Hodoninska below the level of the control. This might be in connection with their utilization of serine for the synthesis of tryptophane and auxin, the concentration of which in resistant plants sharply increases an the 14th day after inoculation (KRATKA and KUDELA, in press). The decrease in tyrosine content might be connected with role in the synthesis of phenolic compounds, melanines, etc., which are known to be involved in plant-defensive reactions (KOSUGE 1969). The greatest changes both in the quantity of synthesized proteins and in the protein pattern, as caused by lucerne bacterial wilt, were recorded in plants of the relatively resistant cultivar on the 20th day and in plants of the susceptible cultivar on the 35th day after inoculation. The experimental results support the hypothesis that the resistance or susceptibility degrees are determined by the rate of plant response to the presence of the pathogen; an immediate activation of alternative metabolic pathways causes resistance, the retarded activation causes susceptibility. This hypothesis is also supported by protein changes, observed in lucerne plants infected with Verticillium albo-atrum (WHITHEY, HEALE and VAUGHAN 1972). Analysis of the infected materials by means of the disc electrophoresis gave evidence of the progressive alternationsin the protein composition in the stems of the susceptible cultivars, whereas the strong initial reaction of resistant cultivar, manifested by the change of the protein structure, turned back towards normal development.
References DE YAY, J. E., CHARUDATTAN, R., and WIMALAJEEWA, D. L. S.: Common antigenic determinants as a possible regulator of host-pathogen compatibility. Amer. Naturalist 106 (1972), 185-194. HECKER, R. J., RUPPEL, E. G., MAAG, G. W., and RASMUSON, D. M.: Amino acids associated with Cercospora leaf spot resistance in sugar beet. Phytopath. Z. 82 (1975),175-181. HEITEFUSS, R.: Fortschritte in der Analyse der Wirt-Parasit-Beziehungen. Angew. Botanik 53 (1979),111-125. KOSUGE, T.: The role of phenolics in host response to infection. Ann. Rev. Phytopath. 7 (1969), 195-222. KREITLOW, K. W.: Infecting of seven-day-old alfalfa seedlings with wilt bacteria through wounded cotyledons. Phytopathology 53 (1963), 800 - 803. KUDELA, V.: Odolnost eeskoslovenskvch odrud vojtesky proti bakterialnfmu vadnutf (Resistance of Czechoslovak lucerne varieties to bacterial wilt). Ochr, Rostl. 7 (XLIV) (1971),169-177. PEGG, G. F., and SEQUEIRA, L.: Stimulation of aeromatic biosynthesis in tobaco plants infected by Pseudomonas solanaceorum: Phytopathology 58 (1968),476-483. RAGGI, V., ZAZZERINI, A., BARBERINI, B., FERBANTI, F., and DBAOLI, R.: Alterations in the composition of free and combined amino acids in susceptible and resistant tomato cultivars infected with Fusarium oyysporum f. lycopersici. Phytopath. Z. 79 (1974), 258 -280.
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RUBIN, B. A.: Problemy biochimii patogeneza i immuniteta rastenij (Biochemical problems of pathogenesis and immunity). In: Immunitet selskoehosjajstvennyeh rastenij k boleznjam i vrediteljam. Moskva (1975), 158-166. SADASIVAN, F. S.: Physiology of wilting plants. In: Biochemische Problerne del' kranken Pflanze. Tagungsbericht Nr. 74. Dtsch. Akad. Landwirtschaftswiss. Berlin (1965), 149 -163. ANDEL, O. M. VAN: Amino acids and plant diseases. Ann. Rev. Phytopathol. 4 (1966), 349-368. ·WHITNEY, P.J., REALE, J. B., and VAUGHAN, J. G.: Protein changes in the vascular wilt disease of lucerne caused by Verticillium albo-atrum R. et B. J. expel'. Bot. 23 (1972), 400-414. Authors' address: Ing. VACLAV KUDELA, CSc., and RNDr. JrRINA KBATKA, CSc., Research Institute for Plant Product.ion CS -161 06 Prague 6·Ruzyne 507, Czechoslovakia.