Phytopath. Z,, 104, 193—201 (1982)© 1982 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / InterCode: PHY2A3

Department of Botany, University of Delhi, Delhi~l 10007, India

Changes in Chloroplast Pigments in Wheat LeavesInfected with. Pucdnia graminis tritici^)

By

HARDEV SINGH, P. D. AGARWAL, G . BHATTACHARYA and INDERJEET SETHI

With 8 figures

Received December 1980

The most typical initial symptom induced by biotrophic parasites is theyellowing of leaves (FARKAS 1978). In such infections there may be a smallincrease in photosynthesis initially but there is a decrease in the later stages.Recent studies point out towards an intact and functional photosyntheticapparatus for some time during pathogenesis (MONTALBINI and BUCHANAN

1974). Since the biotrophic parasites require a contmuous supply of meta-bolites for their own synthetic processes the preservation of structure wasconsidered to be logical. Moreover, noncyclic photophosphorylation has beenfound to be inhibited in infected plants but cyclic photophosphorylation wasunaffected (MONTALBINI and BUCHANAN 1974, MAGYAROSY et al. 1976, seealso DALY 1976, KOSUGE 1978).

The later occurrence of green-islands in higher plant tissues colonised bybiotrophic parasites has been described for a number of diseases (BUSHNELL

1960, ENGELBRECHT 1968, HARDING et al. 1968, ATKIN and NEILANDS 1972).However, investigators have disagreed upon the pattern of green-island for-mation- It has been pointed out by ALLEN (1942) that green-islands in mildewinfected wheat leaves are areas where chlorophyll was destroyed and sub-sequently reformed. In Brassica juncea cotyledons infected by Albugo can-dida, delayed chlorophyll breakdown has been considered as more importantthan chlorophyll resynthesis (HARDING et al. 1968). WANG (1961) reported

1) Presented in a poster form at the 3rd International Congress of Plant Pathology atMunidi in August 1978.

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194 HARDEV SINGH, AGARWAL, BHATTACHARYA and INDERJEET SETHI

that in Pinto bean leaves infected with Uromyces phaseoli, green-island for-mation depends on light conditions after inoculation of the plants.

CoRNU (1881) first referred to the flow of nutrients to the infection sitewhich behaves as a metabolic sink. This view has been supported by severalworkers (sec DEKHUIJZEN 1976). The endogenous cytokinins of rust infectedplants have been reported to play an important role in the transport andaccumulation of nutrients towards and around the infection site. It has beensuggested rhat the high level of cytokinin in the infected leaves may berelated to the appearance of the green islands. However, the origin of theincreased cytokinin activity in the compatible and incompatible combinationsIS not known yet.

Recent studies with the electron microscope showed significant differ-ences in the chloroplast structure of infected and uninfected leaves (MAGYA-

ROSY et al. 1976) and also in the chloroplast of the green island and therapidly senescing parts of the same leaves (CAMP and WHITTINGHAM 1975).The latter workers showed the presence of persistent photosynthetic lamellaein chloroplasts of green-island cells which were almost completely degeneratedin chlorotic cells. In spite of the reduced chloroplast number, the green islandsretained their green colour and this was considered to be due to enoughpigment synthesis and adequate chloroplast lamellae number.

The present work reports the changes observed in the four diloroplast-pigments during disease development in the susceptible and resistant reactions.

Materials and Methods

Plant Materials Used

Triticum aestivum L. cv. Lai Bahadur and Agra Local were used for the susceptiblereactions; and cv. HD 2009, a hybrid of Lerma Rojo 64 A Nainari 60 was used for theresistant reaction. Indian collection of race 122 of Puccinia graminis Pers. f. sp. tritici Erikss.&: Henn. was used as the pathogen.

Plant Culture and Inoculation Procedures

Wheat plants were grown in a greenhouse (temperature 18—29 °C; relative humidity52—62%; 14 h light ioWowed by 10 h darkness). First leaves oi seven days old seedlingswere sprayed with distilled water; uredospores were applied on leaves with the help of aflattened spatula; the leaves were again sprayed with distilled water and incubated in amoist chamber for 24 h.

Extraction of Chloroplast Pigments

Tediniques of WITHAM et al. (1971) were followed (with a minor modification) for theseparation of chloroplast pigments. In each case five strips of the first leaves of the size0.4 /'. 0.5 cm were excised from infected portions every alternate day. These were washedwith distilled water and dried over blotting papers. Extraction of the pigments was startedby grinding the leaf pieces with pestle and mortar in 10 ml of cold 80 % acetone plus a smallamount of CaCOjj. The homogenate was transferred to a separatory funnel with 10 ml ofpetroleum ether. The ether soluble layer was washed thrice with distilled water, and driedwith anhydrous sodium sulphate. The extract was concentrated to 0.5 ml in a water bath.

Changes in Chloroplast Pigments in Wheat Leaves 195

Paper Chromatography

One dimensional ascending chromatograms were run on Whatman No. 1 chromato-graphy paper (size 20 X 10 cm) at room temperature using petroleum ether and benzene (9 :1)as the solvent mixture.

Pigments Determination

After separation, the chlorophyll pigment bands were cut out and were eluted in 10 mlof solvent ether separately. Optical density (O.D.) was measured with the help of Systronic'sSpectrocolorimeter Type 103. O.D. of chlorophyll a was measured at wavelength of 400 nmwhereas for the rest of the pigments it was measured at 440 nm.

Results

I. External morphology of infected leaves

A. Susceptible reaction

Initial chlorosis of the leaves was observed 4—5 days after inoculation.Sori ruptured after 7—8 days and green islands developed 12 days afterinoculation. Senescence of uninfected areas on leaves started after 36 days butthe green islands showed senescence after 40 days. Senescence of control leavescould be observed 45 days after seeding.

B. Resistant reaction

A change in the colour of the infected leaves was observed on the 8thday after inoculation. These chlorotic patches were prominent during 9thand 10th day after inoculation. Sorus formation started on the 12th day butsori ruptured by 14th day. Green islands developed around sori after 18 days.Senescence of uninfected areas on leaves started after 36 days but the greenislands showed senescence after 46 days. Senescence of control leaves started48 days after seeding (42 days after inoculation) and the process was completeon 54th day.

IL Changes in pigments

A. Susceptible reaction

(i) C h l o r o p h y l l a increased in control leaves till 22 days after in-oculation and then decreased till 52 days. In infected plants it increasedslightly till 4 days and then decreased till 10 days. It again increased after14 days and attained a maximum value on 22nd day after which it graduallydeclined obtaining a minimal value which was higher than control on 54thday (Fig. 1).

(ii) C h l o r o p h y l l b showed a trend similar to that of chlorophyll a.In control leaves it increased gradually and then declined whereas in infectedleaves the curve showed a slight dip on the 4th day after infection, to showan upward trend again on the 6th day. The upward trend continued till20 days and then declined gradually. In the uninfected leaves the maximumvalue was attained on the 16th day. The minimum value in control was ob-

196 HARDEV SINGH, AGARWAL, BHATTACHARYA and INDERJEET SETHI

tained on 52nd day which was less than that of the infected leaves obtainedon the 54th day (Fig. 2).

(iii) C a r o t e n e increased in control as well as in infected leaves till 18and 10 days respectively, the maximum value being higher in infected leaves.The minimum value is obtained on 50th day in control and 52nd day in in-fected leaves. Even the minimum value was higher in the infected leaves(Fig. 3).

(iv) X a n t h o p h y l l . In both the control and infected leaves, the maximumvalue was obtained on 22nd day with a gradual decrease till 54th day. Themaximum as well as the minimum values for infected leaves were higher thancontrols (Fig. 4).

0 5

ao 0.1

UnmfectedInfected

CHLOROPHYLL-Q CHLOROPHYLL-b

J I L

30 CO 50a f t e r i n f e c t i o n

0 10 20 30 40 50 0 10 20

d a y s a f t e r i n f e c t i o n days

Figs. 1—4. Changes in the contents of chlorophyll a, b, carotene and xanthophyll in thesusceptible reaction. Each point on the graph represents an average of two repetitions. Mor-phology of the infected leaves in relation to days after infection: initial chlorosis — 4—5 days;eruption of sori — 7—8 days; formation of green islands — 12 days; senescence of un-

infected area — 36 days; yellowing of green islands — 40 days

B. Resistant reaction

(i) C h l o r o p h y l l a increased in uninfected leaves till 29 days afterseeding (22 days after inoculation), and then it gradually decreased till54 days when it became constant (Fig. 5). In infected leaves there was aninsignificant increase in chlorophyll a till 6 days after inoculation and latera decline till 16 days. Afterwards, it started increasing and attained a peak

Changes in Chloroplast Pigments in Wheat Leaves 197

value on 22nd—24th day. It went on decreasing till 52 days and finallybecame constant (Fig. 5). Minimum value for infected leaves was higher thanthat of control leaves.

(ii) C h l o r o p h y l l b increased in control leaves till 25 days after seed-ing (18 days after inoculation). In infected leaves it increased till 16 days afterinoculation. Later in both kinds of leaves it went on decreasing till 56 and58 days after inoculation respectively. The chlorophyll b content in infectedleaves showed higher value than that of control during the period of senes-cence (Fig. 6).

(iii) C a r o t e n e increased in control leaves till 18 days and then grad-ually declined and attained its minimal value on 56th day (Fig. 7). Whereas,in infected leaves it increased till 22 days after inoculation and then went ondecreasing till 60th day. Minimum value of carotene was higher in infectedleaves in contrast to control leaves (Fig. 7).

(iv) X a n t h o p h y l l increased in control as in infected leaves andreached its peak value 18 days after infection in control and 20 days afterinfection in infected leaves (Fig. 8). The peak value was retained for some

UninfectedIn fec ted

CHLOROPHYLL-bCHLOROPHYLL-Q

days a f te r in fec t ion days a f ter in fec t ion

Figs. 5—8. Changes in the contents of chlorophyll a, b, carotene and xanthophyll in theresistant reaction; chlorophyll b does not seem to breakdown during the initial chlorosis. Eachpoint on the graph represents an average of two repetitions. Morphology of the infectedleaves in relation to days after infection: initial chlorosis — 8 days; eruption of sori —14 days; formation of green islands — 18 days; senescence of uninfected area — 36 days;

yellowing of green islands — 46 days

198 HARDFV SINGH, AGARWAL, BHATTACHARYA and INDERJEET SETHI

time in control leaves whereas in infected leaves it showed an immediatedecrease and a gradual decline till 58 days. Minimum value of xanthophyllin infected leaves was slightly higher than control (Fig. 8).

Discussion

In the susceptible and resistant reactions, no significant difference wasobserved in the pattern of increase or decrease in chlorophyll a, and xantho-phyll. However, the pattern of changes in chlorophyll b and carotene wasdifferent in the two reaction types. In the susceptible reaction there wasdegradation of chlorophyll b during initial chlorosis but in the resistantreaction chlorophyll b showed degradation only after reaching the peak value.Carotene contents increased rather quickly in the susceptible reaction. Thusthe initial degradation of chlorophyll b and a quicker increase in carotene maybe used as criteria in characterising the susceptibility of a wheat cultivar tostem rust. However, we do not know the way these may be related to thesusceptibility.

Chlorophyll a and b in susceptible and chlorophyll a in resistant reaction,showed two maxima (Figs. 1,2,4). These observations are in partial agree-ment with those of several workers (WANG 1961, DALY et al. 1962, LIVNE

1964, ZAKI and MIROCHA 1965) who suggested a de novo synthesis of chloro-phyll pigments. The inference drawn by these workers was based upon theirobservation of enhanced physiological activity in the infected leaf tissue. Inthe present investigations this observation could not be confirmed since thepeak values of chlorophyll a and b in the susceptible wheat and that ofchlorophyll a m the resistant wheat were found to be lower than that of thecontrol leaves.

The minimum level of all the pigments during later stages of pathogenesiswas higher in diseased leaves than in the controls of both reaction types.This shows that these pigments were retained in diseased leaves for a longerperiod concluding thereby that the rate of degradation of these pigments waslower in the diseased leaves than in the control leaves as suggested by MUKER-

jEE and SHAW (1962).Unlike chlorophyll a and b, carotenes and xanthophylls showed a steady

increase and then a gradual decline in the diseased leaves with higher amountsof carotenes during early stages in susceptible plants. This can be either due tothe fact that these two pigments are not degraded as the green pigments do,during disease development, or there is an increased synthesis. The third pos-sibility is that the various types of carotenes and other pigments present inthe uredospores of the rust fungi (IRVINE et al. 1954, HOUGEN et al. 1958) maybecome extracted during the experimental procedures. However, this possibilityseems to be remote as the amount of these pigments in spores Is so small that itcannot account for their increased values (IRVINE etal. 1954). Therefore, the in-creased synthesis seems to explain better the higher levels of these two pig-ments in the diseased leaves.

changes in Chloroplast Pigments in Wheat Leaves 199

To know the exact nature and development of green islands more studiesinvolving substances other than pigments are essential and these can be com-pared with green islands induced by exogenously applied chemicals. Thismight give clues as to what is the exact host parasite chemical interaction thatresults in xhe green-islands formation in plants infected with biotrophic fungi.

Summary

Two susceptible wheat cultivars (Agra Local and Lai Bahadur) and aresistance one (HD 2009 — a hybrid of Lerma Rojo 64 X Nainari 60) belong-ing to the Triticum aestivum group; and the race 122 of Puccinia graministritici were used for the study. The initial chlorosis of the infected leavescoincides with an appreciable decrease of chlorophyll a, and a minor decreaseof chlorophyll b only in the susceptible reaction. But with the advance ofthe disease the chlorophyll contents stage a recovery. This seems to be dueto an in situ synthesis of the pigments. During the later regreening of theinfected areas, all the four pigments were found to be at higher levels ascompared to the controls. These results are discussed in relation to the rateof photosynthesis, and the disintegration of chloroplasts; reported in severalprevious studies on plants infected with biotrophic fungi. Susceptibility ofwheat to stem rust may be correlated with the degradation of chlorophyll band a quicker increase in carotene, during initial chlorosis.

Zusammenfassung

Anderungen der Chloroplastenpigmente in Weizenblatterninfiziert mit Paccinia graminis tritici

Fiir die Arbeit wurden zwei anfallige Weizensorten (Agra Local undLai Bahadur) und eine resistente (HD 2009 — ein Hybrid von Lerma Rojo 64X Nainari 60), die zur Gruppe Triticum aestivum gehoren, und Rasse 122von Puccinia graminis tritici benutzt. Die anfangliche Chlorose der infizier-ten Blatter fiel mit einem deutlichen Abfall von Chlorophyll a zusammen undeinem geringeren Abfall von Chlorophyll b, jedoch nur in der anfalligen Re-aktion. Jedoch mit Fortschreiten der Krankheit erfuhr der Chlorophyllgehalteine Erholung. Dies scheint von einer in situ Synthese des Pigments herzuruh-ren. Wahrend des spateren Wiederergriinens der infizierten Stellen wurden allevier Pigmente in groCeren Mengen als bei den Kontrollen gefunden. DieseErgebnisse wurden in Beziehung zur Photosyntheserate und der Disintegrationder Chloroplasten dlskutiert, wie bereits in einigen friiheren Arbeiten iiberPflanzen, die mit biotrophen Pilzen infiziert waren, berichtet wurde. DieAnfalligkeit von Weizen gegenuber Schwarzrost konnte mit dem Abbau vonChlorophyll b und einem schnelleren Anstieg der Carotinoide wahrend deranfanglichen Chlorose korreliert sein.

200 HARDEV SINGH, AGARWAL, BHATTACHARYA and INDERJEET SETHI

One of us (H. S.) is grateful to Prof. R. HEITEFUSS (Gottingen) for his interest andencouragement. We are thankful to Dr. (Mrs.) VANI HARDEV for critically reading themanuscript.

Thanks are due to Mrs. DOROTHEE MENNERICH (Gottingen) for help in the preparationof the illustrations, and to TOM GORE (Victoria, B.C., Canada) for photographic assistance.

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Authors' address: Department of Botany, University of Delhi, Delhi 110007 (India).