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Changes of pectin content in alfalfa plants after inoculation with vascular wilt pathogens
Z bl. M ikro biol, 139 ( 1984), 64 3-648 [Inst it u t e of P lant Product ion, P raha, CSSR]
Changes of Pectin Content in Alfalfa Plants After Inoculation with Vascular 'Wilt Pathogens
Wit h 2 Figu res
Summary Alfa lfa plants at the age of six week s were inoculated wi t h conid ia a n d mycelium sus pension of V erti cillium alboatrum or wit h ce ll sus pen sion of Cory n ebacteri u m in sidiosu m . The r esistant a nd suscep t ible strains, i. e., seed progenies of clones, were in oculated t h rough overground p ar t s or r oot s. Cha nges in pectin content we re de te r m ine d in the overgr oun d p a rts and roots of plants. T h e tota l p ect in conte nt was de te r m ined spec tr o p h ot om et r ica lly . 1t was fo u nd t h at the c ha nges in p ect in content of pl ant s afte r t h e ir ino culatio n wit h th e above m ent ion ed pa thogens were much m or e sign if ica nt in t he r es ist ant p lant s t ha n in t he suscepti ble ones, especia lly at early s ta ges of tho pathogenes is . T he inc rea se or d ecrea se in p ectin content (but n ot t he extent of cha nges) were in flu e nced by the wa y of inoc u lation , es pe c ia lly in t he root s .
Zusammenfassung L uz ernep f la nzen (Medic£/go sativa L .) e ines r esist en ten u nd e ines em p f in d lic hen Stam m es wu rden na ch 6 Woche n m it Cory nebacteri u m insidiosu m od er Vertici llium albo-atrurn b eim p ft, D ie Beimpfung erfolgt.e a n d en o be r ird ische n Organen oder an d on Wurzel n , Der P ekti ngehalt w urde spektrop hotometrisch sowoh l in d e n o b er ird isc hen Pflanzenteilen als a uch in d en \ Vurzeln best im rnt, u nd zw ar 3, 7 und 14 Tage nach d er B ei m pfung . Die Anderung d es P ekt ingeh al te s war in den re aisten ten P fla n zen d eut. licher ausgep ragt. a ls in d en em pf in d lic hen , Er hohung bzw. E rniedrig ung de s P ekt ing eh alt cs, b eso nde rs in d en " ' urzel n , w urde d u rch die Art der B e im p f u ng b eeinflu ut,
.Tn the study of alfalfa plan ts resistance t o fungal and bacterial va scular wilt pa thogens [ Veriicill.ium. albo-atru m. and Corynebacterium insuliosum) ou r attention was focu ssed on some metabolic cha nges within the ino cul ated plan ts . It was discover ed th at after inoculation of the host plants, resistant to Vertici lliurn albo-atrum and Corynebacteriu m insidioeum ; with the above-mentioned pathogens the metabolic p rocesses were stimulated immed iately. Though the abo ve-me n t ioned pathogen s differ in morphological, anatom ical , and biochemical asp ect s, they do not only ca use sim ilar sy mpt oms of di sea se, but they also cau se metaboli c cha nges in the plants ( K RATKA and K UDELA 1979, 1982). On t he basis of result s a lready o btaine d, we cons ider t he rate and t he success ion of cha nges , espec ially at t he ea rly phases of pa t hogenesis, are more imp ortant t ha n the changes t hems elves . These cons iderations agree with WHEELER ' S findings (1975). On the ba sis of external and histological sy mptoms, obtained after testing the resistan ce of alfalfa clon es, and bioc hemical dat a and result s of t he radiotracing measur emen t s, we cam e to t he concl us ion t hat t he cell wa ll plays an important role in the protective mechanism of t he host ag a inst the va scular wilt pa thogens (KRATKA, K UDE LA 1979, K UDELA 1982, K UDE LOVA, K UDELA et a l. 1978, 1980).
644
J.
KRATKA
and J.
CHLUMSKA
ALBERSHEIM et al. (1969) suggested on the basis of their previous investigations the following conclusions: 1. Pathogenesis of the parasite is, in many cases, the result of the relation between the pathogen and the hydrocarbons of the host, which is determined by the ability of pathogens to produce the enzymes, destroying the cell wall of the host. 2. Production of these enzymes decides on starting a successful infection. The plants respond actively to the presence of pathogens 3. Corresponding metabolic reactions are going on at the considered host - pathogen complex differently. The presented results are basing on our previous papers and summarize the observations on the changes of pectin content in young alfalfa plants after inoculation with Verticillium albo-atrum or Oorynebacterium insidiosum,
Material and Methods We experimented with three alfalfa (JJIedicaqo sativa L.) strains, i.e., seed progenies of clones. Strain No. 340 Strain No. 354 Strain No. 321 -
resistant to C. insidiosum and V. albo-atrum; susceptible to C. insidiosum, susceptible to V. albo-utrum,
Alfalfa plants were inoculated at t.he age of six weeks with conidia and mycelium a suspension of Verticillium alboatrum or with cell suspension of Corynebacterium insidioeurn, The inoculation was carried out in two ways:
1. The overground parts of plants were cut off with scissors, immersed in inoculum. Inoculum was applied to fresh cuts immediately after cutting. 2. The roots and overground parts were shortened and plunged into suspension of inoculum for the period of 16 hrs. After inoculation the plants were put into specially equipped vessels, filled with nutrient solution. Sampling of plants for biochemical analyses was carried out on the day of inoculation and on the third, seventh, and fourteenth day after inoculation. The pectin content was determined spectrophotometrically at A = 520 nm, using the earbasol method (McCREANY and MCCOMB 1952)_ Total pectin content was determined in ovorground parts and roots separately. All analytical determinations were carried out in six replicates. The pectin amount was determined in p,g galacturonic acid· ml- 1 extract.
Results and Discussion In overground parts and roots of resistant plants, inoculated through overground parts with both pathogens, the decrease of pectin content was significant three days after inoculation. The decrease is evident even, at the fourteenth day after inoculation (Fig. 1). In susceptible plants, inoculated through overground parts, the decrease of pectin was not significant at the third day after inoculation. A more significant difference in comparison with the control plants was not visible up to the seventh and especially up to the fourteenth day (Fig. 1). A significant decrease of pectin content in overground parts took place in resistant plants, inoculated through the roots with both pathogens, especially at the third day. The decrease of pectin content was still notiecable at the seventh day, but there was no difference in pectin content between control and inoculated plants at the fourteenth day. Changes in pectin content in the roots were different. On the third day a significant increase of pectin content was found in comparison with the control. The increase was evident even at the fourteenth day (Fig. 2).
Pectin Content in Alfalfa Plants After Inoculation
645
VERTICILLIUM albo-atrum roun roots • 340
L j
90
60
30
o
01230123 VERTICILLIUM albo- atrum roo roots
354
321
90
60
30
o
012
3
0 1 2
3
012
3
0 1 2
3
Fig. 1. Changes of pectin content in alfalfa plants, inoculated through overground parts with Corynebacterium insidiosum and Verticilliun» albo-atrum, 340 Strain of alfalfa plants, resistant to Corynebacterium insidiosum and Verticillium albo-atrum, 354 Strain of alfalfa plants, susceptible to Corynebacterium insidioeum, 321 Strain of Alfalfa plants, susceptible to Verticillium albo-atrum, A Pectin content (flog galacturonic acid, ml- 1 extract). 0, 1, 2, 3 Day of inoculation, third, seventh, and fourteenth day after inoculation. 0---0 Pectin content in inoculated plants, Pectin content in control plants.
e---e
646
J. KRATKA and J. CHLUMSKA
3
o 12
3
012
3
012
3
012
3
CORYNEBACTERIUM insidiosum round
roots
over
roots
354
321
30
012
3
0
2
3
0 1 2
3
0 1 2
3
Fig. 2. Changes of pectin content in alfalfa plants, inoculated through roots with Corynebacterium insidiosum and Verticillium albo-atrum, 340 Strain of alfalfa plants, resistant to Corynebacterium insidiosum and Verticillium albo-atrum, 354 Strain of alfalfa plants, susceptible to Corynebacterium insidiosum. 321 Strain of alfalfa plants, susceptible to Verticillium albo-atrum, A Pect.in content (flg galacturonic acid, ml- 1 extract). 0, 1, 2, 3 Day; of inoculation, third, seventh, and fourteenth day after inoculation. Pectin content in inoculated plants, --~ Pectin content in control plants.
Pectin Content in Alfalfa Plants After Inoculation
647
In the susceptible plants, inoculated through the roots, no essential changes were found either in the overground parts or roots. On the basis of the obtained results it can be stated that the infection of young alfalfa plants with fungal or bacterial pathogens (Verticillium albo-atrum or Oorynebacterium insidiosum) causes changes in pectin content. These changes are significant, especially in the resistant plants after inoculation with either pathogen. In the susceptible plants these changes proceed less significantly and more slowly. The extent of the changes - decrease or increase -are influenced by the inoculation method. Different ways of inoculation were used, since the inoculated plants were used for some experiments (e.g., observation with labelled elements). Surprisingly, considerable changes in the content of pectin, found after inoculation of resistant plants (in most cases a decrease), encourage further investigation concerning the way of pectin role in the formation of the resistance alfalfa plants against vascular wilt pathogens. In recent years the activity of polygalacturonases and pectinlyases of young alfalfa plants, inoculated with the above vascular wilt pathogens was studied (KRATKA and KUDELA 1979, 1980, 1982). However, comparing the obtained results, no time correlation with enzyme activities and content of pectins was exhibited. ALBERSHEIM et al. (1969) summarized the results of their investigation and the results by other authors as well. The following conclusions were drawn: Hydrocarbons of the host plants in contact with pathogens control the synthesis of enzymes degrading polysaccharides of the cell wall. Minute modifications of the composition, structure or accessibility of the host plant carbohydrates may change quantity and type of enzymes produced with the pathogens. Thus, the presence of an enzyme produced by pathogens does not need to cause the degradation of the cell wall. These facts contribute to considerations on participation of pectinolytic enzymes in changes of pectin content in the plants after inoculation with vascular wilt pathogens. LAMPORT (1970) deals with the problems concerning the synthesis of pectin constituents of the cell wall. On the basis of STODDARD'S and NORTHCOTE'S (1967) results, he confirms that individual cell wall polysaccharides are not necessarily inert after the entry bound into the wall. The obtained results raise a number of questions on the possible participation of the host cell wall in plant resistance to fungal and bacterial pathogens. In our investigation we are concerned with: 1. Determination of the degree of pathogen pectinolytic enzymes, autolysis or inhibition of synthesis of pectin compounds of the cell wall in the changes of pectins after the inoculation of alfalfa plants with vascular wilt pathogens. 2. The possibility of single cell wall components or their precursors, participating in the complex of biochemical processes in alfalfa plants, resistant to vascular wilt pathogens.
648
J. KHATKA and .J. CHLUMSKA, Pectin Content in Alfalfa Plants After Inoculation
References ALBERSHEIM, P., JONES, T. M., and ENGLISH, P. D.: Biochemistry of the cell wall in relation to infective processes. Ann. Rev. PhytopathoJ. 7 (1969),171-194. KRATKA, J., and KUDELA, V.: Biochemical changes in alfalfa plants inoculated with Veticillium albo-atrum. Phytopath. Z. 100 (1979), 289-299. - Research report, VORV-Ruzyne (1980), 44pp. - Changes in alfalfa plants metabolism induced by Corynebacterium insidiosum. Phytopath. Z. 101 (1982), 352-362. KlTDELOVA, A., KUDELA, V., BERG~IANOVA, E., TAIMR, L., and HANKER, I.: Dynamika pfijmu 45Ca u vojtesku infikovane Corynebacterium insidiosum (McCulloch) Jensen. Sbornik referabu VII. OS konf. ochr, rostJ. (VII. Czechoslovak Plant Protection Conference), Nitra (1978), 143. - and HANKER, I.: Vliv baktorialnfho vadnutina distrihuci foliarne aplikovaneho 45Ca v rostlinach vojtesky. Sbornik referatu VIII. OS konf. ochr. rostl. (VIII. Czechoslovak Plant Protection Conference), Praha (1980). 207-208. MCCREADY, R. M., and MCCOMB, E. A.: Extraction and determination of total pectin materials in fruits. Analyt. Chern. 24 (1952), 1986-1988. Authors' address: RNDr JIIUNA KRATKA, CSc., Ing. JITKA CHLUMSKA, Research Institute of Crop Production, Section of Plant Protection, CS - 161 06 Praha-6, Drnovska 507.
Changes of Pectin Content in Alfalfa Plants After Inoculation with Vascular 'Wilt Pathogens
Wit h 2 Figu res
Summary Alfa lfa plants at the age of six week s were inoculated wi t h conid ia a n d mycelium sus pension of V erti cillium alboatrum or wit h ce ll sus pen sion of Cory n ebacteri u m in sidiosu m . The r esistant a nd suscep t ible strains, i. e., seed progenies of clones, were in oculated t h rough overground p ar t s or r oot s. Cha nges in pectin content we re de te r m ine d in the overgr oun d p a rts and roots of plants. T h e tota l p ect in conte nt was de te r m ined spec tr o p h ot om et r ica lly . 1t was fo u nd t h at the c ha nges in p ect in content of pl ant s afte r t h e ir ino culatio n wit h th e above m ent ion ed pa thogens were much m or e sign if ica nt in t he r es ist ant p lant s t ha n in t he suscepti ble ones, especia lly at early s ta ges of tho pathogenes is . T he inc rea se or d ecrea se in p ectin content (but n ot t he extent of cha nges) were in flu e nced by the wa y of inoc u lation , es pe c ia lly in t he root s .
Zusammenfassung L uz ernep f la nzen (Medic£/go sativa L .) e ines r esist en ten u nd e ines em p f in d lic hen Stam m es wu rden na ch 6 Woche n m it Cory nebacteri u m insidiosu m od er Vertici llium albo-atrurn b eim p ft, D ie Beimpfung erfolgt.e a n d en o be r ird ische n Organen oder an d on Wurzel n , Der P ekti ngehalt w urde spektrop hotometrisch sowoh l in d e n o b er ird isc hen Pflanzenteilen als a uch in d en \ Vurzeln best im rnt, u nd zw ar 3, 7 und 14 Tage nach d er B ei m pfung . Die Anderung d es P ekt ingeh al te s war in den re aisten ten P fla n zen d eut. licher ausgep ragt. a ls in d en em pf in d lic hen , Er hohung bzw. E rniedrig ung de s P ekt ing eh alt cs, b eso nde rs in d en " ' urzel n , w urde d u rch die Art der B e im p f u ng b eeinflu ut,
.Tn the study of alfalfa plan ts resistance t o fungal and bacterial va scular wilt pa thogens [ Veriicill.ium. albo-atru m. and Corynebacterium insuliosum) ou r attention was focu ssed on some metabolic cha nges within the ino cul ated plan ts . It was discover ed th at after inoculation of the host plants, resistant to Vertici lliurn albo-atrum and Corynebacteriu m insidioeum ; with the above-mentioned pathogens the metabolic p rocesses were stimulated immed iately. Though the abo ve-me n t ioned pathogen s differ in morphological, anatom ical , and biochemical asp ect s, they do not only ca use sim ilar sy mpt oms of di sea se, but they also cau se metaboli c cha nges in the plants ( K RATKA and K UDELA 1979, 1982). On t he basis of result s a lready o btaine d, we cons ider t he rate and t he success ion of cha nges , espec ially at t he ea rly phases of pa t hogenesis, are more imp ortant t ha n the changes t hems elves . These cons iderations agree with WHEELER ' S findings (1975). On the ba sis of external and histological sy mptoms, obtained after testing the resistan ce of alfalfa clon es, and bioc hemical dat a and result s of t he radiotracing measur emen t s, we cam e to t he concl us ion t hat t he cell wa ll plays an important role in the protective mechanism of t he host ag a inst the va scular wilt pa thogens (KRATKA, K UDE LA 1979, K UDELA 1982, K UDE LOVA, K UDELA et a l. 1978, 1980).
644
J.
KRATKA
and J.
CHLUMSKA
ALBERSHEIM et al. (1969) suggested on the basis of their previous investigations the following conclusions: 1. Pathogenesis of the parasite is, in many cases, the result of the relation between the pathogen and the hydrocarbons of the host, which is determined by the ability of pathogens to produce the enzymes, destroying the cell wall of the host. 2. Production of these enzymes decides on starting a successful infection. The plants respond actively to the presence of pathogens 3. Corresponding metabolic reactions are going on at the considered host - pathogen complex differently. The presented results are basing on our previous papers and summarize the observations on the changes of pectin content in young alfalfa plants after inoculation with Verticillium albo-atrum or Oorynebacterium insidiosum,
Material and Methods We experimented with three alfalfa (JJIedicaqo sativa L.) strains, i.e., seed progenies of clones. Strain No. 340 Strain No. 354 Strain No. 321 -
resistant to C. insidiosum and V. albo-atrum; susceptible to C. insidiosum, susceptible to V. albo-utrum,
Alfalfa plants were inoculated at t.he age of six weeks with conidia and mycelium a suspension of Verticillium alboatrum or with cell suspension of Corynebacterium insidioeurn, The inoculation was carried out in two ways:
1. The overground parts of plants were cut off with scissors, immersed in inoculum. Inoculum was applied to fresh cuts immediately after cutting. 2. The roots and overground parts were shortened and plunged into suspension of inoculum for the period of 16 hrs. After inoculation the plants were put into specially equipped vessels, filled with nutrient solution. Sampling of plants for biochemical analyses was carried out on the day of inoculation and on the third, seventh, and fourteenth day after inoculation. The pectin content was determined spectrophotometrically at A = 520 nm, using the earbasol method (McCREANY and MCCOMB 1952)_ Total pectin content was determined in ovorground parts and roots separately. All analytical determinations were carried out in six replicates. The pectin amount was determined in p,g galacturonic acid· ml- 1 extract.
Results and Discussion In overground parts and roots of resistant plants, inoculated through overground parts with both pathogens, the decrease of pectin content was significant three days after inoculation. The decrease is evident even, at the fourteenth day after inoculation (Fig. 1). In susceptible plants, inoculated through overground parts, the decrease of pectin was not significant at the third day after inoculation. A more significant difference in comparison with the control plants was not visible up to the seventh and especially up to the fourteenth day (Fig. 1). A significant decrease of pectin content in overground parts took place in resistant plants, inoculated through the roots with both pathogens, especially at the third day. The decrease of pectin content was still notiecable at the seventh day, but there was no difference in pectin content between control and inoculated plants at the fourteenth day. Changes in pectin content in the roots were different. On the third day a significant increase of pectin content was found in comparison with the control. The increase was evident even at the fourteenth day (Fig. 2).
Pectin Content in Alfalfa Plants After Inoculation
645
VERTICILLIUM albo-atrum roun roots • 340
L j
90
60
30
o
01230123 VERTICILLIUM albo- atrum roo roots
354
321
90
60
30
o
012
3
0 1 2
3
012
3
0 1 2
3
Fig. 1. Changes of pectin content in alfalfa plants, inoculated through overground parts with Corynebacterium insidiosum and Verticilliun» albo-atrum, 340 Strain of alfalfa plants, resistant to Corynebacterium insidiosum and Verticillium albo-atrum, 354 Strain of alfalfa plants, susceptible to Corynebacterium insidioeum, 321 Strain of Alfalfa plants, susceptible to Verticillium albo-atrum, A Pectin content (flog galacturonic acid, ml- 1 extract). 0, 1, 2, 3 Day of inoculation, third, seventh, and fourteenth day after inoculation. 0---0 Pectin content in inoculated plants, Pectin content in control plants.
e---e
646
J. KRATKA and J. CHLUMSKA
3
o 12
3
012
3
012
3
012
3
CORYNEBACTERIUM insidiosum round
roots
over
roots
354
321
30
012
3
0
2
3
0 1 2
3
0 1 2
3
Fig. 2. Changes of pectin content in alfalfa plants, inoculated through roots with Corynebacterium insidiosum and Verticillium albo-atrum, 340 Strain of alfalfa plants, resistant to Corynebacterium insidiosum and Verticillium albo-atrum, 354 Strain of alfalfa plants, susceptible to Corynebacterium insidiosum. 321 Strain of alfalfa plants, susceptible to Verticillium albo-atrum, A Pect.in content (flg galacturonic acid, ml- 1 extract). 0, 1, 2, 3 Day; of inoculation, third, seventh, and fourteenth day after inoculation. Pectin content in inoculated plants, --~ Pectin content in control plants.
Pectin Content in Alfalfa Plants After Inoculation
647
In the susceptible plants, inoculated through the roots, no essential changes were found either in the overground parts or roots. On the basis of the obtained results it can be stated that the infection of young alfalfa plants with fungal or bacterial pathogens (Verticillium albo-atrum or Oorynebacterium insidiosum) causes changes in pectin content. These changes are significant, especially in the resistant plants after inoculation with either pathogen. In the susceptible plants these changes proceed less significantly and more slowly. The extent of the changes - decrease or increase -are influenced by the inoculation method. Different ways of inoculation were used, since the inoculated plants were used for some experiments (e.g., observation with labelled elements). Surprisingly, considerable changes in the content of pectin, found after inoculation of resistant plants (in most cases a decrease), encourage further investigation concerning the way of pectin role in the formation of the resistance alfalfa plants against vascular wilt pathogens. In recent years the activity of polygalacturonases and pectinlyases of young alfalfa plants, inoculated with the above vascular wilt pathogens was studied (KRATKA and KUDELA 1979, 1980, 1982). However, comparing the obtained results, no time correlation with enzyme activities and content of pectins was exhibited. ALBERSHEIM et al. (1969) summarized the results of their investigation and the results by other authors as well. The following conclusions were drawn: Hydrocarbons of the host plants in contact with pathogens control the synthesis of enzymes degrading polysaccharides of the cell wall. Minute modifications of the composition, structure or accessibility of the host plant carbohydrates may change quantity and type of enzymes produced with the pathogens. Thus, the presence of an enzyme produced by pathogens does not need to cause the degradation of the cell wall. These facts contribute to considerations on participation of pectinolytic enzymes in changes of pectin content in the plants after inoculation with vascular wilt pathogens. LAMPORT (1970) deals with the problems concerning the synthesis of pectin constituents of the cell wall. On the basis of STODDARD'S and NORTHCOTE'S (1967) results, he confirms that individual cell wall polysaccharides are not necessarily inert after the entry bound into the wall. The obtained results raise a number of questions on the possible participation of the host cell wall in plant resistance to fungal and bacterial pathogens. In our investigation we are concerned with: 1. Determination of the degree of pathogen pectinolytic enzymes, autolysis or inhibition of synthesis of pectin compounds of the cell wall in the changes of pectins after the inoculation of alfalfa plants with vascular wilt pathogens. 2. The possibility of single cell wall components or their precursors, participating in the complex of biochemical processes in alfalfa plants, resistant to vascular wilt pathogens.
648
J. KHATKA and .J. CHLUMSKA, Pectin Content in Alfalfa Plants After Inoculation
References ALBERSHEIM, P., JONES, T. M., and ENGLISH, P. D.: Biochemistry of the cell wall in relation to infective processes. Ann. Rev. PhytopathoJ. 7 (1969),171-194. KRATKA, J., and KUDELA, V.: Biochemical changes in alfalfa plants inoculated with Veticillium albo-atrum. Phytopath. Z. 100 (1979), 289-299. - Research report, VORV-Ruzyne (1980), 44pp. - Changes in alfalfa plants metabolism induced by Corynebacterium insidiosum. Phytopath. Z. 101 (1982), 352-362. KlTDELOVA, A., KUDELA, V., BERG~IANOVA, E., TAIMR, L., and HANKER, I.: Dynamika pfijmu 45Ca u vojtesku infikovane Corynebacterium insidiosum (McCulloch) Jensen. Sbornik referabu VII. OS konf. ochr, rostJ. (VII. Czechoslovak Plant Protection Conference), Nitra (1978), 143. - and HANKER, I.: Vliv baktorialnfho vadnutina distrihuci foliarne aplikovaneho 45Ca v rostlinach vojtesky. Sbornik referatu VIII. OS konf. ochr. rostl. (VIII. Czechoslovak Plant Protection Conference), Praha (1980). 207-208. MCCREADY, R. M., and MCCOMB, E. A.: Extraction and determination of total pectin materials in fruits. Analyt. Chern. 24 (1952), 1986-1988. Authors' address: RNDr JIIUNA KRATKA, CSc., Ing. JITKA CHLUMSKA, Research Institute of Crop Production, Section of Plant Protection, CS - 161 06 Praha-6, Drnovska 507.