Gas-exchange, plastid pigments and some other physiological parameters were determined in bean (Phaseolus vulgaris L. local populations) leaves naturally infected by Xanthomonas campestris pv.phaseoli (Smith) Dye, and Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie, and in healthy leaves (control).
It was established that infected leaves had lower both plastid pigments content and photosynthetic activity as well as lower yield and quality of produce.Проучено е влиянието на естественото инфектиране с Xanthomonas campestris pv.phaseoli (Smith) Dye, и
Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie, върху листния газообмен, пластидните пигменти и някои други физиологични показатели в листата на фасула.
Установено е, че инфектираните листа се характеризират както с понижено съдържание на пластидните пигменти и фотосинтетичната активност, така и с намален добив и каческтво на продукцията

MALGOJATA BEROVA

NEVENA STOEVA

PETER CHAVDAROV

TZVETELINA STOILOVA

ZLATKO ZLATEV

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ORIGINAL PAPER57Volume 8 (2007) No. 1 (57-62)PHYSIOLOGICAL CHANGES IN BEAN (PHASEOLUS VULGARIS L.) LEAVES, INFECTED BY THE MOST IMPORTANT BEAN DISEAФИЗИОЛОГИЧНИ ИЗМЕНЕНИЯ В ЛИСТАТА ОТ ФАСУЛ (PHASEOLUS VULGARIS L.), ИНФЕКТИРАНИ С ИКОНОМИЧЕСКИ ВАЖНИ БОЛЕСТИ ПО ТАЗИ КУЛТУРАMALGOJATA BEROVA1*, NEVENA STOEVA1, ZLATKO ZLATEV1, TZVETELINA STOILOVA2, PETER CHAVDAROV21 Agricultural University, Department of Plant Physiology and Biochemistry, 12 Mendeleev Str., 4000 Plovdiv, Bulgaria, tel: +359 32 654408, e-mail: maberova@abv.bg2 Institute of Plant Genetic Resources - Sadovo, BulgariaABSTRACTGas-exchange, plastid pigments and some other physiological parameters were determined in bean (Phaseolus vulgaris L. local populations) leaves naturally infected by Xanthomonas campestris pv.phaseoli (Smith) Dye, and Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie, and in healthy leaves (control).It was established that infected leaves had lower both plastid pigments content and photosynthetic activity as well as lower yield and quality of produce.Keywords: Phaseolus vulgaris L., Xanthomonas campestris pv.phaseoli (Smith) Dye (Xcp), Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie (Psp), leaf gas exchange, chlorophyll ﬂuorescence, plastid pigments, peroxidase activity, water content, elements of productivityРЕЗЮМЕПроучено е влиянието на естественото инфектиране с Xanthomonas campestris pv.phaseoli (Smith) Dye, и Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie, върху листния газообмен, пластидните пигменти и някои други физиологични показатели в листата на фасула. Установено е, че инфектираните листа се характеризират както с понижено  съдържание на пластидните пигменти и фотосинтетичната активност, така и с намален добив и каческтво на продукцията.   Ключови думи: Phaseolus vulgaris L., Xanthomonas campestris pv.phaseoli (Smith) Dye (Xcp), Pseudomonas syringae pv.phaseolicola (Bukholder) Young, Dye et Wilkie (Psp), листен газообмен, хлорофилна флуоресценция, пластидни пигменти, пероксидазна активност, водно съдържание, елементи на продуктивността58 Journal of Central European Agriculture Vol 8 (2007) No 1MALGOJATA BEROVA, NEVENA STOEVA, ZLATKO ZLATEV, TZVETELINA STOILOVA, PETER CHAVDAROVINTRODUCTIONCommon bean (Phaseolus vulgaris L.) is a legume crop frequently and severely attacked by common (caused by Xanthomonas campestris pv. phaseoli (Smith) Dye) and halo (caused by Pseudomonas syringae pv.phaseolicola (Bulholder) Young, Dye et Wilkie) blight [1, 10, 12]. Symptoms of these diseases are very similar and it is not always possible to separate the losses caused by common blight and those due to halo blight, since they frequently occur together in the same ﬁeld and on the same plants [13]. The pathogens attack all aerial plant parts, including leaf petioles, pods and seeds, but the characteristic symptoms of chlorotic borders around the necrotic lesions (Xcp) and red-brown necrotic spots (Psp) are more severe and conspicuous on leaves of susceptible cultivars. Xсp and Psp could be powerful stress factors inducing signiﬁcant anatomy-morphological and physiology-biochemical disorders in plant organisms [3, 5, 6, 15, 16]. The latter could have a negative effect on yields and quality of produce. The existence of Xcp and Psp in all parts of the world indicate the necessity to identify in detail these pathogens in Bulgaria in order to help the breeding program select the right parental materials. MATERIALS AND METHODSThe experiments were carried out in the years 2004-2005’ at the ﬁeld microtrials with bean plants (Phaseolus vulgaris L.) - four Bulgarian landraces from our collection (IPGR-Sadovo).The leaf gas exchange parameters – net photosynthetic rate (PN), transpiration rate (E), stomatal conductance (gs) were determined with a portable photosynthetic system LCA-4 (Analytical Development Company Ltd., Hoddesdon, England). Measurements were made under a light intensity of 1400 μmol m-2 s-1 PAR, at a temperature of 25±28 ºC, an external CO2 concentration of 450 μmol mol-1 and relative air humidity of 65-70%. Parameters of chlorophyll ﬂuorescence were determinated with a photosynthesis yield analyser MINI-PAM (H. Walz, Germany) after 30 min of dark adaptation of the plants, as described by Schreiber et al [11]. Chlorophyll (Chl) and carotenoids (Car) were extracted with 80% water acetone. The pigments were determined spectrophotometrically (Specol 11, K. Zeiss, Jena, Germany) after centrifugation of extract at 3000 rpm for 5 min [17] and calculated according Lichtenthaler and Wellburn formulae [8].Peroxidase activity (PA; EC 1.11.1.7) was determined according to Herzog and Fahimi [4]. The material was homogenized with 0.05M Tris-glycine buffer, pH 8.3, containing 17% sucrose (m/v). One relative PA unit (U) is equal to ∆A 470 g-1 min-1.  Relative water content (RWC) was determined according to Morgan [9].The analyses were made on leaves of the same physiological age in three replications.The agroclimatic conditions for bean production in Table 1. Leaf gas exchange parameters in healthy and naturally infected by Xanthomonas campestris pv. phaseoli (Smith) Dye, and Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, Bulgarian landraces of Phaseolus vulgaris L.; PN - net photosynthetic rate [µmol (CO2) m-2 s-1]; E - transpiration rate [mmol m-2 s-1]; gs - stomatal conductance [mol m-2 s-1]; * P < 0.05; ** P < 0.01;������� 1. ������������ �� ������� ��������� � ��������� � ���������� ����������� � Xanthomonas campestris pv. phaseoli (Smith) Dye, � Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, ��������� ������� ����� Phaseolus vulgaris L.; PN - ������� �� ������������� [µmol (CO2) m-2 s-1];E - ������������ �� �������������� [mmol m-2 s-1]; gs - ������� ����������� [mol m-2 s-1];* P < 0.05; ** P < 0.01;Parameters Landraces Variants PN E gscontrol (healthy) 19.83�0.22 2.57�0.15 0.16�0.01 2000E-01 naturally infected 12.69�0.32**  2.86�0.12* 0.18�0.02* control (healthy) 21.81�0.30 4.22�0.10 0.22�0.04 95E-05 naturally infected 17.88�0.36* 4.48�0.10* 0.22�0.02 control (healthy) 20.45�0.22 3.15�0.11 0.20�0.01 99E-059 naturally infected 15.13� 0.40** 3.22�0.10 0.22�0.01* control (healthy) 20.98�0.16 3.46�0.09 0.18�0.03 93E-012 naturally infected 15.94�0.21** 3.67�0.12* 0.21�0.01** PHYSIOLOGICAL CHANGES IN BEAN (PHASEOLUS VULGARIS L.) LEAVES, INFECTEDBY THE MOST IMPORTANT BEAN DISEA59J. Cent. Eur. Agric. (2007) 8:1, 57-62the years of carrying out the experiment were medium favourable to favourable. In 2004 and 2005, there were strong rains in July, but temperature was higher than the mean value for the last 10 years. The rains at ﬁrst helped vegetation but then complicated it as were favourable to advent of diseases.The data were statistically analysed. The differences between control and other variants were evaluated according to Student’s t-criterion [18].RESULTS AND DISCUSSIONThe effects of common and halo bacterial blight on the photosynthetic rates of healthy and diseased bean leaves were determined by gas exchange analysis (Table 1).The results showed that the leaf gas exchange rate in the infected leaves of all investigated forms was changes. The net photosynthetic rate was strongly suppressed - by 18% (95E-01) to 36% (2000E-01). There is a consensus that a decrease of photosynthesis is Table 2. Chlorophyll a and b, and carotenoids content in healthy and naturally infected by Xanthomonas campestris pv. phaseoli (Smith) Dye, and Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, bulgarian landraces of Phaseolus vulgaris L.; Chl. a, �hl. b, Car. [mg g-1 d. m.]; * P < 0.05; ** P < 0.01; ������� 2. ���������� �� �������� a � b, � ����������� � ��������� � ���������� ����������� �Xanthomonas campestris pv. phaseoli (Smith) Dye, � Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, ��������� ������� ����� Phaseolus vulgaris L.; ��. a, ��. b, ���. [mg g-1 d. m.]; * P < 0.05; ** P < 0.01; Parameters    Landraces Variants Chl � Chl b Car control (healthy) 3.34�0.03 1.84�0.05 2.19�0.02 2000E-01 naturally infected 1.74�0,02** 0.94�0,01** 1.74�0,01** control (healthy) 4.20�0.07 2.03�0.01 2.63�0.04 95E-05 naturally infected 2.73�0.06** 1.46�0.04** 2.49�0.02* control (healthy) 3.40�0.04 1.89�0.05 2.78�0.07 99E-059 naturally infected 2.14�0.03** 1.23�0.02** 2.08�0.04** control (healthy) 4.11�0.06 1.78�0.04 2.80�0.01 93E-012 naturally infected 2.79�0.05** 1.32�0.03** 1.96�0.02** Table 3. Effects of common (Xanthomonas campestris pv. phaseoli (Smith) Dye) and halo (Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie) bacterial blight on some physiological parameters in bean leaves; Fv/Fm - variable to maximum fluorescence ratio, PA - peroxidase activity [U]; RWC - relative water content [%]; * P < 0.05; ** P < 0.01;������� 3. ������� �� Xanthomonas campestris pv. phaseoli (Smith) Dye), � Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie ����� ����� ������������ ��������� � ������� �� �����;Fv/Fm - ��������� ����������/���������� �������������, PA - ������������ ��������� [U]; RWC -����������� ����� ���������� [%]; * P < 0.05; ** P < 0.01;Parameters Landraces Variants Fv/Fm PA RWC control (healthy) 0.796�0.03 625�12 70�1.5 2000E-01 naturally infected 0.604�0.02** 900�18**  49�0.9**  control (healthy) 0.825�0.01 720�15 74�1.4 95E-05 naturally infected 0.700�0.02* 979�14**  55�0.6**  control (healthy) 0.781�0.05 595�18 69�1.1 99E-059 naturally infected 0.609�0.03** 892�19**  47�1.2**  control (healthy) 0.793�0.02 686�21 72�2.0 93E-012 naturally infected 0.634�0.05** 974�16**  49�1.2**  60 Journal of Central European Agriculture Vol 8 (2007) No 1MALGOJATA BEROVA, NEVENA STOEVA, ZLATKO ZLATEV, TZVETELINA STOILOVA, PETER CHAVDAROVattributed to both stomatal and non-stomatal limitations [14]. As it was showed in Table 1 transpiration rate and stomatal conductance in the infected leaves were higher than controls. That’s why it can be assumed that mainly non-stomatal limitation of photosynthesis take place. These have been attributed to damages in the primary photochemical (inhibited functional activity of PSII and reduced amount of photosynthetic pigments) and biochemical processes [7]. Xcp and Psp induced changes in content of photosynthetic pigment (Table 2). A statistically signiﬁcant decrease by 24% (95E-05) to 48% (2000E-01) in chlorophyll a content was detected. There was a similar effect on the content of chlorophyll b. We suggest that the reduced total chlorophyll content in infected plants was due on the activation of its enzymatic degradation.The stronger decrease of chlorophyll content as compared to that of carotenoids provoked changes in their interrelations.  The Fv/Fm ratio is a parameter for the potential PSII efﬁciency in the photochemical   reactions. It is know that in healthy leaves this ratio is in the range of 0.75-0.85 [2]. The signiﬁcant Fv/Fm decrease in the infected plants (by about 20%) was indicative of serious PSII disturbances (Table 3).Under stress plants possess several tissue antioxidant enzymes for protection against the potentially cytotoxic forms of activated oxygen species, among them peroxidase (PA) being most important. The results showed, that the PA activity in infected leaves was increased by 36 to 50% above the control.  The changes in the relative water content (RWC) of all investigated forms were signiﬁcant. RWC in infected Table 4. Elements of productivity of healthy and naturally infected by Xanthomonas campestris pv. phaseoli(Smith) Dye, and Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, Bulgarian landraces of Phaseolus vulgaris L.; * P < 0.05; ** P < 0.01;������� 4. �������� �� ��������������� � ��������� � ���������� ����������� � Xanthomonas campestris pv. phaseoli (Smith) Dye, � Pseudomonas syringae pv. phaseolicola (Bukholder) Young, Dye et Wilkie, ��������� ������� ����� Phaseolus vulgaris L.; * P < 0.05; ** P < 0.01;Parameters Landraces Variants Number of pods per plant Number of seeds per plant 100 seeds weight [g]  control (healthy) 5,3�0.25 11.7�1.0 32,4�1.2 2000E-01 naturally infected 2,0�0.08**  4.0�0.05**   23,9�2.3**   control (healthy) 15,1�1.2 37.7�2.8 32,9�1.6 95E-05 naturally infected 8,5�0.61**  21.5�2.2**  28,7�2.5*  control (healthy) 11,5�0.79 22.3�4.1 38,2�1.9 99E-059 naturally infected 6,2�0.52**  12.3�1.9** 31,3�2.2* control (healthy) 13,9�0.62 29.4�2.6 42,4�2.8 93E-012 naturally infected 6,1�0.40** 15.4�1.3**  37,3�2.1* leaves was by 26% to 32% lower comparison to healthy such. Xcp and Psp gave the negative effect on productivity of plants (Table 4). All studies forms had lower number of pods and seeds per plant. The weight of 100 seeds was strongly reduced as a result of slight growth of them. The best, in terms of having the least difference in comparison to the control, was 95E-05 population.On the basis of the physiological parameters studied the following conclusion can be made: The studies forms had differ in their resistance to common and halo blight. Some of them had better and some worse tolerance to diseases. On the basis of the data obtained we could specify 95E-05 as population with a higher level of resistance to common and halo blight comparison to the other three. ACKNOWLEDGEMENTSWe thank Ms. M. Necheva for her technical assistance during the experiments.REFERENCES[1] Ariyarathe H., Coyne D., Vidaver A., Eskridge K., Pathogenic variation in Pseudomonas syringae pv. phaseolicola strains on common beans, Bean Improvement Cooperative (2001) 44: 129-130.[2] Bolhar-Nordenkampf H., Oquist G., Chlorophyll ﬂuorescence as a tool in photosynthesis research, in: Hall D., Scurlock J., Bolhar-Nordenkampf H., Leegood R., Long S. (Eds), Photosynthesis and production in a changing environment: A ﬁeld and laboratory manual, PHYSIOLOGICAL CHANGES IN BEAN (PHASEOLUS VULGARIS L.) LEAVES, INFECTEDBY THE MOST IMPORTANT BEAN DISEA61J. Cent. Eur. Agric. (2007) 8:1, 57-62Chapman and Hall, London, 1993, pp. 193-205.[3] Gorlenko M., Bacterial diseases of plants, M. Kolos, 1977, (Ru). [4] Herzog V., Fahimi H., A new sensitive colorimetric assay for peroxidase using 3,3’-diaminobenzidine as hydrogen donor, Anal. Biochem. (1973) 55: 554-562.[5] Izrailskiy V., Bacterial diseases of plants, M. 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(1999) 154: 753-758.[15] Smith E., McLaughlin W., Characterization of extracelluar enzymes and exopolysacharides produced by Xanthomonas campestris pv. phaseoli and some studies of their role in common blight disease in bean, Proceeding of the Fourth Conference of Pure and Applied Sciences, Mona, January, 1999, pp. 12-14.[16] Stancheva Y., General pathology of plants, Soﬁa-Moskva, 2004, (Bg).[17] Welschen R., Bergkotte M., Ecophysiology, Handbook of methods, Department of Plant Ecology and Evolutionary Biology, The Netherlands, 1994.[18] Zaprjanov Z., Marinkov E., Methodology of Experiments and Biometry, Hr. Danov, Plovdiv, 1979, (Bg).

ФИЗИОЛОГИЧНИ ИЗМЕНЕНИЯ В ЛИСТАТА ОТ ФАСУЛ (PHASEOLUS VULGARIS L.), ИНФЕКТИРАНИ С ИКОНОМИЧЕСКИ ВАЖНИ БОЛЕСТИ ПО ТАЗИ КУЛТУРА

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