Early photosynthetic responses of sweet orange plants infected with Xylella fastidiosa

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1 Physiological and Molecular Plant Pathology 62 (23) Early photosynthetic responses of sweet orange plants infected with Xylella fastidiosa Rafael V. Ribeiro a, *, Eduardo C. Machado b, Ricardo F. Oliveira a Laboratório de Fisiologia de Plantas em Condições de Estresse, Departamento de Ciências Biológicas, Escola Superior de Agricultura Luiz de Queiroz, CP 9, Universidade de São Paulo, , Piracicaba/SP, Brazil b Centro de Ecofisiologia e Biofísica, Instituto Agronômico de Campinas, CP 28, , Campinas/SP, Brazil Accepted 5 March 23 Abstract Photosynthetic processes of sweet orange plants [Citrus sinensis (L.) Osbeck cv. Pera] infected with Xylella fastidiosa were investigated. Simultaneous measurements of leaf gas exchanges and chlorophyll a fluorescence were taken in healthy and infected leaves before development of symptoms. The photosynthetic oxygen evolution response to different light intensities and curves of photosynthesis induction were carried out in leaf discs extracted from the same leaves used for gas exchange measurements. Infected plants showed decreased CO 2 assimilation, transpiration and stomatal conductance. No differences between healthy and infected intact leaves regarding chlorophyll a fluorescence parameters were observed, except for the potential quantum yield of photosystem II which was higher in infected plants. Values of photosynthetic oxygen evolution, effective quantum yield of photosystem II, apparent electron transport rate, and photochemical fluorescence quenching were higher in healthy leaf discs than in infected ones. Our results suggest that low photosynthetic rates of non-symptomatically sweet orange leaves infected with X. fastidiosa were caused by low stomatal conductance, biochemical alterations of photosynthetic machinery and increase in alternative electron sinks. q 23 Elsevier Ltd. All rights reserved. Keywords: Citrus sinensis; Xylella fastidiosa; Photosynthesis; Citrus variegated chlorosis; Chlorophyll fluorescence 1. Introduction Citrus variegated chlorosis (CVC), reported in Brazil in 1987 [39], is one of the major threats to the Brazilian citrus industry [1]. The CVC, caused by the bacterium Xylella fastidiosa Wells, is a vascular disease of sweet orange that imposes large reductions in crop production [38]. X. fastidiosa also affects other economically important crops, such as peaches, grapevines, alfalfa, almond, Abbreviations: A, CO 2 assimilation; A m, photosynthetic oxygen evolution; AES, alternative electron sinks; Ci, intercellular CO 2 concentration; CVC, citrus variegated chlorosis; DF=F m, effective quantum efficiency of photosystem II; E, transpiration; ETR, apparent electron transport rate; fco 2, quantum efficiency of CO 2 fixation; F v /F m, potential quantum efficiency of photosystem II; g s, stomatal conductance; NPQ, non-photochemical fluorescence quenching; PPFD, photosynthetic photon flux density; PSII, photosystem II; qp, photochemical fluorescence quenching; R; dark respiration rate. * Corresponding author. Tel.: þ ; fax: þ address: rvribeir@esalq.usp.br (R. V. Ribeiro). ragweed, periwinkle, elm, oak, mulberry, sycamore, and coffee [34,37]. Plants infected with X. fastidiosa exhibit leaf chlorosis that develops to necrosis, leaf wilting similar to those of water stress and low quality fruits [38]. In general, the low productivity of infected plants could be a consequence of low photosynthetic rates that result from necrotic leaf lesions [29], an imbalance in nutrient and growth regulators, toxins [15,19] and low stomatal conductance [9,19,29]. There is a considerable disagreement regarding the pathogenicity mechanisms of X. fastidiosa. The main hypotheses of pathogenicity proposed are: (i) dysfunction of water-conducting system, (ii) phytotoxin production, and (iii) growth regulator imbalance [19]. There is some evidence to support hypothesis (i) [19,29]. However, the low number of occluded xylem vessels and the lack of visible leaf wilting in grapevines with Pierce s disease have indicated that the chlorosis and necrosis may also be caused by toxins [15,38] /3/$ - see front matter q 23 Elsevier Ltd. All rights reserved. doi:1.116/s (3)38-9

2 168 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) Leaf water potential, CO 2 assimilation, transpiration and stomatal conductance declined in infected sweet orange plants [29]. Reduction in photosynthesis of infected plants is related to water stress, caused by decreased sap flow rates [29]. Leaves without visible symptoms also manifested reductions in photosynthetic capacity suggesting that other physiological mechanisms were affected [29], such as biochemical and/or photochemical reactions of photosynthesis and/or hormonal imbalance, possibly caused by toxins or the bacterium itself. In peaches and grapevines infected with X. fastidiosa, decreases in photosynthesis were associated with toxins and hormonal imbalance [2,15]. Changes in levels of hormones, amino acids (proline), and carbohydrates have been observed in infected grapevines [15]. However, infected sweet orange trees did not present similar pattern of the one observed in grapevines (unpublished work). The main objective of this study was to determine how the photosynthetic process is affected by X. fastidiosa before visible symptoms become apparent, by measuring leaf gas exchanges, chlorophyll a fluorescence and photosynthetic oxygen evolution. 2. Materials and methods The measurements of leaf gas exchanges, chlorophyll a fluorescence, and photosynthetic oxygen evolution were carried out in 9 month-old seedlings of sweet orange Citrus sinensis (L.) Osbeck cv. Pera], grown in 3 L plastic pots with soil mixture (one-half soil, one-quarter sand, onequarter cow dung, and nitrogen-phosphate-potassium fertilizer), under greenhouse conditions (maximal and minimum air temperatures of 42 and 188C respectively, minimum RH of 3%, maximal irradiance about 18 mmol m 22 s 21, and photoperiod between 13 4 and 1 6 h). The cv. Pera was chosen because it is the most important sweet orange cv. in Brazil. A nutrient solution was applied periodically according to Van Raij et al. [42] to ensure that no nutrient deficiency could limit growth. Irrigation was provided every morning, until soil saturation. Weekly pesticide applications prevented any occurrence of insects or additional disease. X. fastidiosa inoculation was according to Almeida et al. [1]. Needle inoculation was done by probing five times with a number insect pin through a 2-mL drop of bacterial suspension into three positions on the stem of the seedlings. There were two lots, the first with four healthy plants and the second with four infected plants. All plants were analyzed by the polymerase chain reaction (PCR) [31] or by isolation and culture in periwinkle wilt-gelrite solid medium [1,18] for bacterium detection. Infected plants did not show any visible symptoms of CVC, such as leaf chlorosis, necrosis and wilting, although the PCR and isolation results have confirmed the presence of X. fastidiosa. Measurements started seven months after inoculation with X. fastidiosa immediately after its detection. Healthy and infected sweet orange Pera seedlings were moved to a plant growth chamber (E-15, Conviron, Winnipeg, Canada), under a temperature regime of 35/28C (day/night), 14 h photoperiod, photosynthetic photon flux density (PPFD) of 6 mmol m 22 s 21 and air vapor-pressure deficit of 1 kpa. The temperature regime of 35/28C was chosen because it was shown to be optimal for the growth of citrus seedlings. The measurements of CO 2 and H 2 O vapor fluxes were taken at 358C in mature and fully expanded (about 6 monthold) leaves located in stem third medium, with an infrared gas analyzer (LI-64, LICOR, Lincoln, NE USA) in open system. CO 2 assimilation (A), transpiration (E), stomatal conductance ðg s Þ; and intercellular CO 2 concentration ðc i Þ were calculated by the LI-64 data analysis program, which uses the Von Caemmerer and Farquhar [43] general gas exchange formula. The environmental conditions of the LI-64 leaf chamber were the same as for those of the plant growth chamber E-15. A, E, g s and C i were stored when the coefficient of total data variation was less than 5%. As measurements were taken in leaves without visible symptoms of CVC, the effects of reduction in the photosynthetically active area were excluded. The respiration rate ðrþ was measured during the dark period. The measurements of gas exchanges and chlorophyll a fluorescence were taken simultaneously, with a modulated fluorometer (FMS1, Hansatech, King s Lynn, UK) adapted to the gas analyses sensor head of the LI-64. Maximal ðf m Þ and basal ðf o Þ fluorescence yield were measured in dark-adapted (3 min) leaves, whereas steady-state ðf s Þ and maximal ðf m Þ fluorescence were sampled in light-adapted state [41]. Variable fluorescence yield was determined in dark-adapted ðf v ¼ F m 2 F o Þ and in light-adapted ðdf ¼ F m 2 F s Þ states. F o was the basal fluorescence after photosystem I excitation by far-red light. The parameters calculated were: potential ðf v =F m Þ and effective ðdf=f m Þ quantum efficiency of photosystem II (PSII) [14], photochemical (qp) and non-photochemical (NPQ) fluorescence quenching, and apparent electron transport rate (ETR), whereas qp ¼½ðF m 2 F s Þ=ðF m 2 F o ÞŠ; NPQ ¼ ½ðF m 2 F m Þ=F m Š; and ETR ¼ðPPFD DF=F m 5 84Þ [3,21]. For the calculation of ETR, 5 was used as the fraction of excitation energy distributed to PSII, and 84 was used as the fractional light absorbance. Alternative electron sinks (AES) were calculated as the relation between the effective quantum efficiency of PSII ðdf=f m Þ and quantum efficiency of CO 2 fixation (fco 2 ), as AES ¼ðDF=F m =fco 2 Þ [24]. fco 2 was calculated according to Edwards and Baker [1]: f CO 2 ¼½ðAþRÞ=ðPPFD 84ÞŠ; where PPFD is the light reaching the leaf (6 mmol m 22 s 21 ) and 84 is the fractional light absorbance. Photosynthetic oxygen evolution ða m Þ was measured at 358C, with a leaf disc oxygen electrode (LD2/3 leaf

3 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) chamber, Hansatech, King s Lynn, UK). PPFDs were provided by external light source (LS3, Hansatech, King s Lynn, UK) and A m values were recorded in PC (Oxygraph measurement system v. 2 22, Hansatech, King s Lynn, UK). The CO 2 saturant concentration in the chamber was generated by 2 cm 3 of carbonate/bicarbonate buffer solution (1 M, 1:19 v/v) [7,8]. In these conditions, photorespiration and CO 2 flow limitation to chloroplasts through stomata are almost eliminated and it is possible to analyze photosynthetic process without that limitation [8,44]. Immediately after excision of a 1 cm 2 leaf disc from attached leaves (same leaves of gas exchange measurements), the leaf discs were enclosed in LD2/3 leaf chamber, with a wet felt disc to maintain the leaf water status constant during photosynthesis measurements [44]. Light response curves of A m were obtained varying PPFD values from 33 to 1121 mmol m 22 s 21, at 358C. Before photosynthesis measurements, leaf discs were kept in dark conditions for 3 min, and then submitted to low irradiance (128 mmol m 22 s 21 ) for 1 min for photosynthesis induction [44]. After this period, PPFD was decreased to 33 and a gradual increase was done until reaching a maximum value of 1121 mmol m 22 s 21 (33, 58, 74, 92, 12, 232, 343, 531, 791 and 1121 mmol m 22 s 21 ). Leaf temperature was controlled using a water bath (MA-127, Marconi, Piracicaba, SP Brazil) and monitored with a copper-constantan thermocouple (AWG 24, Omega Eng., Stamford, CT USA) attached to the under surface of the leaf disc. Simultaneous measurements of A m ; DF=F m ; ETR, qp, and NPQ were taken during photosynthesis induction [4, 44], using the FMS1 fluorometer attached to LD2/3 leaf chamber. These measurements were taken during the induction phase of light response curve (128 mmol m 22 s 21 ). Leaf discs were kept for 3 min in darkness at 358C, before measurements. After this, discs were exposed to light and A m ; DF=F m ; qp, NPQ, and ETR were sampled at 15 s intervals, for 6 min. The experiment was arranged in a completely randomized design with three or four replicates. All results were subjected to analysis of variance procedures. Tukey s test at the 5 probability level was employed to determine the statistical significance between measured parameters of healthy and infected plants. 3. Results and discussion 3.1. Gas exchanges and chlorophyll a fluorescence in attached leaves X. fastidiosa infected plants had lower A, E, and g s than healthy ones ðp, 5Þ (Table 1). These values may be related to a lower leaf water content [29] caused by the partial blockage of xylem vessels by X. fastidiosa [11]. Sweet orange trees infected with X. fastidiosa also showed a lower stomatal conductance, with statistical difference between healthy and non-symptomatically infected leaves [29]. Xylella fastidiosa caused decreases of 35, 29, and 37% in g s ; E, and A, respectively. The decrease in A was probably due to partial stomatal closure. According to Khairi and Hall [2] and Nobel [32] the same reduction in g s would reduce E more than A. Thus, low g s did not appear to explain completely the decrease in photosynthesis of infected plants. C i remained statistically constant (Table 1). At first sight, there is CO 2 available for photosynthesis, but the calculation of C i might not be reliable when CO 2 and water vapor fluxes are low, as happens in infected plants. High values of C i could be attributed to patchy stomatal closure and the increased importance of cuticular transpiration as stomata close, causing an over estimation of C i and thus masking any stomatal effect on the reduction of A [5]. The chlorophyll a fluorescence parameter F v =F m in infected plants was higher than in healthy ones ðp, 5Þ (Table 2). Environmental stresses that affect PSII efficiency usually lead to F v =F m decrease [22]. Reduction in F v =F m have been related to photoinhibition under natural conditions [25,3]. Heat or cold stresses can also lead to a lowering PSII quantum efficiency [17]. As the blockage of xylem vessels [11] causes water deficit within the plant body [33,36], perhaps the highest F v =F m values of infected plants are due to a higher PSII potential efficiency caused by water stress. Water stress could strengthen lipid-psii protein interactions, through changes in the lipid composition of the thylakoid membranes in plants under water stress [12,17,35]. X. fastidiosa did not affect ETR, qp, and NPQ (Table 2). Under natural conditions (21% of O 2 and 37% of CO 2 ), decreases in A, not followed by decreases in qp, indicate that molecular oxygen could serve as an electron acceptor, Table 1 Effects of X. fastidiosa infection on CO 2 assimilation (A), transpiration (E), stomatal conductance ðg s Þ; intercellular CO 2 concentration ðc i Þ; and respiration (R) of sweet orange Pera attached leaves Plants Parameters* A(mmol m 22 s 21 ) E (mmol m 22 s 21 ) g s (mol m 22 s 21 ) C i (mmol mol 21 ) R (mmol m 22 s 21 ) Healthy 4 1 ^ 34 a 1 81 ^ 32 a 87 ^ 12 a 232 ^ 38 a 3 7 ^ 71 a Infected 2 53 ^ 27 b 1 29 ^ 7 b 57 ^ 7 b ^ 56 4 a 3 38 ^ 51 a * Data represent means ^ SD (n ¼ 4 from separate plants). Values in each column followed by the same letter are not significantly different (at P, 5Þ using the Tukey test.

4 17 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) Table 2 Effects of X. fastidiosa infection on the potential ðf v =F m Þ and effective ðdf=f m Þ quantum efficiency of photosystem II, apparent electron transport rate (ETR), photochemical (qp), and non-photochemical (NPQ) fluorescence quenching, and alternative electron sinks (AES) of sweet orange Pera attached leaves Plants Parameters* F v =F m DF=F m ETR qp NPQ AES Healthy 775 ^ 3 b 632 ^ 15 a ^ 3 9 a 9 ^ 1 a 37 ^ 8 a 43 5 ^ 3 2 b Infected 795 ^ 2 a 632 ^ 9 a ^ 2 3 a 89 ^ 1 a 26 ^ 11 a 56 6 ^ 5 8 a * Data represent means ^ SD (n ¼ 4 from separate plants). Values in each column followed by the same letter are not significantly different (at P, 5Þ using the Tukey test. re-oxidize the plastoquinone pool and maintain high qp values [4]. Our results suggest the possibility of the existence of a leaf alternative sink activity. Even with similar values of DF=F m ; ETR, qp, and NPQ (Table 2), CO 2 assimilation was lower in infected plants than in healthy ones (Table 1). Usually, some electrons produced by the photochemical process are required for alternative sinks. This alternative electron transport is accelerated when the carbon fixation is limited [23,26,27]. It is related to the role of plant protection, such as the action of antioxidants on active oxygen species [13]. The alternative sinks for electrons are photorespiration and reduction of molecular oxygen [24]. If more electrons are used in these processes, the ratio between effective quantum efficiency of PSII and quantum efficiency of CO 2 fixation tends to increase (higher AES) [24]. AES was 23% higher in infected plants than in healthy ones (Table 2). Thus, it is suggested that more electrons were diverted to photorespiration and/or Mehler reaction than to photosynthetic processes [1,24]. This result was supported by low values of A and the same R of infected plants (Table 1). Photorespiration could be a mechanism to avoid photoinhibition, mainly in C 3 plants [16], dissipating excess ATP and NADPH, producing internal CO 2 to maintain Rubisco activity and consuming strong oxidants such as H 2 O 2, during catalasis [28]. the hypothesis that the photosynthetic mechanism may not only be constrained by lower g s : When compared to healthy plants, X. fastidiosa infected plants showed lower DF=F m and ETR during the full induction period ðp, :5Þ (Fig. 2). According to Maxwell and Johnson [3], DF=F m is the proportion of absorbed energy used in photochemistry and it is related to achieved efficiency. DF=F m and ETR showed similar patterns during photosynthesis induction, supporting the higher oxygen evolution of healthy plants in relation to infected ones (Fig. 2). The presence of X. fastidiosa caused changes in quenching coefficients (Fig. 3). qp was higher in healthy plants at the start of induction ðp, 5Þ: Higher qp indicates that more fluorescence was quenched by the photochemical process, or that more energy was quenched in the primary photochemical reactions [22]. NPQ had a marked increase ðp, 5Þ from 12 s after the start of induction in infected plants (Fig. 3), indicating that the X. fastidiosa infection induced higher energy dissipation due to heat generation and thylakoid membrane energization [4]. Sweet orange plants infected with X. fastidiosa dissipated excess of energy by increasing NPQ under 3.2. Light response curves of oxygen evolution, photosynthesis induction and quenching analysis in leaf discs The highest values of A m were found in healthy plants, mainly when light saturation of healthy plants was higher than 4 mmol m 22 s 21 PPFD ðp, 5Þ: X. fastidiosa caused a mean decrease of 31 4% in A m ðp, 5Þ; but the quantum efficiency of photosynthetic oxygen evolution, as measured by the initial linear region of the light curves (until 1 mmol m 22 s 21 ) of healthy and infected plants was not statistically different (Fig. 1). In infected plants light saturation occurred around 343 mmol m 22 s 21 ðp, 5Þ: As the method employed to quantify A m eliminated the influence of stomata and suppressed photorespiration [44], the lowest values of A m found in infected plants support Fig. 1. Light response curve of photosynthetic oxygen evolution in leaf discs of healthy and infected sweet orange plants. Each point represents the mean ^ SD ðn ¼ 3Þ:

5 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) Fig. 3. Photochemical (a) and non-photochemical (b) fluorescence quenching of healthy and infected sweet orange leaf discs during photosynthesis induction period. Each point represents the mean ^ SD ðn ¼ 3Þ: Fig. 2. Photosynthetic oxygen evolution (a), effective quantum efficiency of PSII (b) and apparent electron transport rate (c) of healthy and infected sweet orange leaf discs during photosynthesis induction period. Each point represents the mean ^ SD ðn ¼ 3Þ: non-photorespiratory conditions, where the protective effects of this mechanism were eliminated. The responses of NPQ were contrary to qp. The energy dissipation during the photochemical process tended to increase while the dissipation through non-photochemical process decreased with the induction of photosynthesis. This observation confirms the existence of a mechanism of competition between these two processes (Fig. 3). A m was greater in healthy plants, likewise in the light response curve, during the photosynthesis induction period, suggesting that some restriction, direct or indirect, of photochemistry and/or biochemistry determined the decrease in A m of plants infected with X. fastidiosa. We suggest that the lowest values of A m in infected plants were probably caused by a biochemical injury of photosynthesis as evidenced in the non-photorespiratory condition. Machado et al. [29] reported that decreases in A, E, g s, and leaf water potential in infected sweet orange plants were associated with water deficit. It is proposed that X. fastidiosa causes water deficit by the obstruction of xylem vessels [11, 29,33,36]. The gum deposition in xylem vessels of grapevine with Pierce s disease and peaches with phony disease is the first internal symptom [11]. This deposition, associated with the increase in tylose formation, occurs before external symptoms become apparent. Thus, one of the probable causes of decreases in A and g s in infected plants without visible symptoms is the obstruction in xylem vessels caused by gum and tyloses. Sweet orange trees with CVC showed about 56 % of reduction on daily sap flow when cultivated at field conditions [33]. Thus, the lowest values of g s and A in infected plants would be caused by water stress promoted by X. fastidiosa. Also, the decrease in A of plants infected with pathogens could be caused by the loss of photosynthetically active leaf

6 172 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) area [6]. However, changes in photosynthesis of plants infected with X. fastidiosa were detected in leaf tissues without disease symptoms, indicating that X. fastidiosa damaged the plants before the development of visible symptoms. These results were also reported by Machado et al. [29]. Besides stomatal effects, photosynthesis may further be affected by leaf necrotic lesions, nutritional imbalance, and the possible pathogen toxin production [6,9]. We argue that X. fastidiosa induces some kind of injury to the biochemical reactions of photosynthesis, since measurements of oxygen evolution eliminated the effect of stomata. The biochemical injury could be caused by toxins produced, or an indirect consequence of possible plant responses to pathogen infection. The low number of obstructed xylem vessels and the lack of any visible leaf wilting of grapevines showing Pierce s disease led to the hypothesis that toxins produced by bacteria are responsible for leaf chlorosis and necrosis [15]. However, the existence of these toxins have not been proven and the influence of these toxins on photosynthetic mechanisms is unclear. The symptomatological and biophysical characteristics of peaches infected with X. fastidiosa did not support the hypothesis that symptoms were entirely the result of a xylem vessel obstruction [2]. Besides affecting the photosynthetic mechanism through stomatal closure and injuries in biochemical reactions, X. fastidiosa also promoted decreased photosynthesis by increasing AES, such as photorespiration, by an increase in the Rubisco oxygenase activity [23,26,27]. The highest AES values of infected plants determined with chlorophyll a fluorescence measurements were not different from those obtained from healthy plants, as indicated in Table 2. Thus, in conditions when photorespiration is present the photochemical apparatus is protected. When photorespiration is not operational, infected plants dissipate excessive energy by heat formation, as indicated by higher values of NPQ (Fig. 3). In conclusion, our results suggest that the lower photosynthetic rates of non-symptomatically sweet orange leaves infected with X. fastidiosa were caused by low stomatal conductance, biochemical injuries to the photosynthetic machinery and an increase in alternative electron sinks. Acknowledgements R.V.R. is grateful to FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil, proc. 98/ and /2325-4) and E.C.M. is grateful to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) for fellowships granted. We thank Rosangela C. Marucci and Prof. João R. S. Lopes for X. fastidiosa isolates and detection tests. References [1] Almeida RPP, Pereira EF, Purcell AH, Lopes JRS. Multiplication and movement of a citrus strain of Xylella fastidiosa within sweet orange. Plant Disease 21;85: [2] Andersen PC, French WJ. Biophysical characteristics of peach trees infected with phony peach disease. Physiological and Molecular Plant Pathology 1987;31:25 4. [3] Bilger W, Schreiber U, Bock M. Determination of the quantum efficiency of photosystem II and non-photochemical quenching of chlorophyll fluorescence in the field. Oecologia 1995;12: [4] Bolhàr-Nordenkampf HR, Öquist GO. Chlorophyll fluorescence as a tool in photosynthesis research. In: Hall DO, Scurlock JMO, Bolhàr- Nordenkampf HR, Leegood RC, Long SP, editors. Photosynthesis and Production in a Changing Environment: A Field and Laboratory Manual. London: Chapman and Hall; p [5] Cornic G. Drought stress inhibits photosynthesis by decreasing stomatal aperture not by affecting ATP synthesis. Trends in Plant Science 2;5: [6] Daly JM. The carbon balance of diseased plants: changes in respiration, photosynthesis and translocation. In: Heitefuss R, Williams PH, editors. Physiological plant pathology, Encyclopedia of Plant Physiology, 4. Berlin: Springer-Verlag; p [7] Da Matta FM, Maestri M, Mosquim PR, Barros RS. Photosynthesis in coffee (Coffea arabica and C. canephora) as affected by winter and summer conditions. Plant Science 1997;128:43 5. [8] Delieu T, Walker DA. Polarographic measurement of photosynthetic oxygen evolution by leaf discs. New Phytologist 1981;89: [9] Duniway JM. Water status and imbalance. In: Heitefuss R, Williams PH, editors. Physiological plant pathology, Encyclopedia of Plant Physiology, 4. Berlin: Springer-Verlag; p [1] Edwards GE, Baker NR. Can carbon dioxide assimilation in maize leaves be predicted accurately from chlorophyll fluorescence analysis. Photosynthesis Research 1993;37: [11] Esau K. Anatomic effects of the viruses of pierce s disease and phony peach. Hilgardia 1948;18: [12] Ferrari-Iliou R, Pham Thi AT, Vieira da Silva J. Effect of water stress on the lipid and fatty acid composition of cotton (Gossypium hirsutum) chloroplasts. Physiologia Plantarum 1984;62: [13] Foyer CH, Descourvières P, Kunert KJ. Protection against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant, Cell and Environment 1994;17: [14] Genty B, Briantais JM, Baker NR. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 1989;99: [15] Goodwin PH, Devay JE, Meredith CP. Roles of water stress and phytotoxins in the development of pierce s disease of the grapevine. Physiological and Molecular Plant Pathology 1988;32:1 15. [16] Hall DO, Rao KK. Photosynthesis: studies in biology. Cambridge: Cambridge University Press; [17] Havaux M. Stress tolerance of photosystem II in vivo. Antagonistic effects of water, heat, and photoinhibition stresses. Plant Physiology 1992;1: [18] Hill BL, Purcell AH. Multiplication and movement of Xylella fastidiosa within grapevine and 4 other plants. Phytopathology 1995;85: [19] Hopkins DL. Xylella fastidiosa: xylem-limited bacterial pathogen of plants. Annual Review of Phytopathology 1989;27: [2] Khairi MMA, Hall AE. Temperature and humidity effects on net photosynthesis and transpiration of citrus. Physiologia Plantarum 1976;36: [21] Krall JP, Edwards GE. Relationship between photosystem II activity and CO 2 fixation in leaves. Physiologia Plantarum 1992;86:18 7.

7 R.V. Ribeiro et al. / Physiological and Molecular Plant Pathology 62 (23) [22] Krause GH, Weis E. Chlorophyll fluorescence and photosynthesis: the basics. Annual Review of Plant Physiology and Plant Molecular Biology 1991;42: [23] Laisk A, Rasulov BH, Loreto F. Thermoinhibition of photosynthesis as analyzed by gas exchange and chlorophyll fluorescence. Russian Journal of Plant Physiology 1998;45: [24] Leipner J, Fracheboud Y, Stamp P. Effect of growing season on the photosynthetic apparatus and leaf antioxidative defenses in two maize genotypes of different chilling tolerance. Environmental and Experimental Botany 1999;42: [25] Long SP, Humphries S, Falkowski PG. Photoinhibition of photosynthesis in nature. Annual Review of Plant Physiology and Plant Molecular Biology 1994;45: [26] Loreto F, Tricoli D, Di Marco G. On the relationship between electron transport rate and photosynthesis in leaves of the C4 plant Sorghum bicolor exposed to water stress, temperature changes and carbon metabolism inhibition. Australian Journal of Plant Physiology 1995; 22: [27] Loreto F, Di Marco G, Tricoli D, Sharkey TD. Measurements of mesophyll conductance, photosynthetic electron transport and alternative electron sinks of field grown wheat leaves. Photosynthesis Research 1994;41: [28] Lüttge U, Kluge M, Bauer G. Botanique: traité fondamental. Paris: Lavoisier: Tec and Doc; [29] Machado EC, Quaggio JA, Lagôa AMMA, Ticelli M, Furlani PR. Gas exchange and water relations of orange trees with citrus variegated chlorosis. Brazilian Journal of Plant Physiology 1994;53 7. in Portuguese. [3] Maxwell K, Johnson GN. Chlorophyll fluorescence a practical guide. Journal of Experimental Botany 2;51: [31] Minsavage GV, Thompson CM, Hopkins DL, Leite RMVBC, Stall RE. Development of a polymerase chain-reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 1994;84: [32] Nobel PS. Physicochemical and environmental plant physiology. San Diego: Academic Press; [33] Oliveira RF, Machado EC, Marin FR, Medina CL. Sap flow rates and stomatal conductance of sweet orange Pêra (Citrus sinensis L. Osb.). IX International Society Of Citriculture Congress. Orlando: ISC; 2. p [34] Paradela Filho MR, Sugimori MH, Ribeiro IJA, Garcia Jr A, Beretta MJG, Harakawa R, Machado MA, Laranjeira FF, Neto JR, Beriam LOS. Constatação de Xylella fastidiosa em cafeeiro no Brasil. Summa Phytopathologica 1997;23:46 9. [35] Prabha C, Arora YK, Wagle DS. Phospholipids of wheat chloroplasts and its membranes under water stress. Plant Science 1985;38: [36] Purcell AH, Hopkins DL. Fastidious xylem-limited bacterial plant pathogens. Annual Review of Phytopathology 1996;34: [37] Raju BC, Wells JM. Diseases caused by fastidious xylem-limited bacteria and strategies for management. Plant Disease 1986;7: [38] Rossetti V. Clorose variegada dos citros (CVC). In: Rodrigues O, Viégas F, Pompeu J Jr, Amaro AA, editors. Clorose Variegada Dos Citros (CVC). Campinas: Fundação Cargill; p [39] Rossetti V, Garnier M, Bové JM, Beretta MJG, Teixeira ARR, Quaggio JA, De Negri JD. Présence de bactéries dans le xyleme d orangers atteints de chlorose variégée, une nouvelle maladie des agrumes au Brésil. Comptes Rendus Academy Scientific 199;31: [4] Schreiber U, Bilger W. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: Tenhunen JD, Catarino FM, Lange OL, Oechel WC, editors. NATO ASI Series, Series G; Plant Response to Stress. Berlin: Springer-Verlag; p [41] Van Kooten O, Snel JFH. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthesis Research 199;25: [42] Van Raij B, Cantarella H, Quaggio JA, Furlani AMC. Recomendações de adubação e calagem para o Estado de São Paulo. Campinas: Instituto Agronômico and Fundação IAC; [43] Von Caemmerer S, Farquhar GD. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 1981;153: [44] Walker DA. Use of the oxygen electrode and fluorescence probes in simple measurements of photosynthesis. Chichester: Oxygraphics Ltd; 199.