Physiological and Molecular Plant Pathology #2002) 60, 185±195 doi:10.1006/pmpp.2001.0388, available online at http://www.idealibrary.com on

Water relations, histopathology and growth of common bean Phaseolus vulgaris L.) during pathogenesis of Macrophomina phaseolina under drought stress

NETZAHUALCOÂ YOTL MAYEK-PEÂ REZ*, ROBERTO GARCIÂ A-ESPINOSA, CAÂ NDIDO LOÂ PEZ-CASTANÄ EDA, JORGE A. ACOSTA-GALLEGOS and JUNE SIMPSON 1Departamento de QuõÂmica, Centro de Ciencias BaÂsicas, Universidad AutoÂnoma de Aguascalientes, Universidad 940, Aguascalientes, 20100 MeÂxico, 2Colegio de Postgraduados, Carretera MeÂxico-Texcoco km. 36.5, Montecillo, Texcoco 56230 MeÂxico, 3Campo Experimental Valle de MeÂxico, Instituto Nacional de Investigaciones Forestales, AgrõÂcolas y Pecuarias /INIFAP), Apartado Postal 10, Chapingo, 56230 MeÂxico and 4Departamento de IngenierõÂa GeneÂtica, CINVESTAV-Unidad Irapuato, Apartado Postal 629, Irapuato, 36500 MeÂxico

#Accepted for publication 14 February 2002)

Alterations in water relations, growth and histopathology caused by Macrophomina phaseolina, causal agent of charcoal rot, and drought stress were characterized in Phaseolus vulgaris L. under controlled conditions. P. vulgaris cultivars BAT 477 and TLP 19 #resistant) and Pinto UI-114 and Rio Tibagi #susceptible) were cultivated under irrigation and drought stress conditions in infested or uninfested pots with a highly virulent isolate of M. phaseolina. Drought stress showed higher negative e€ects than M. phaseolina on water relations, vegetative growth and histopathology in P. vulgaris. Drought stress decreased transpiration rate, water potential, osmotic potential, turgor potential, relative water content, leaf area and dry weight of all vegetative structures of P. vulgaris. Drought stress increased charcoal rot development and stomatal resistance, and increased the association among physiological and growth characteristics and charcoal rot development. M. phaseolina invaded between epidermal cells of BAT 477 and Pinto UI-114 hypocotyls. The fungus infected cortex tissues, vascular cylinder, and pith cells of Pinto UI-114, but only epidermal and parenchyma cells of BAT 477. Typical symptoms caused by M. phaseolina were found to be associated with damage caused by the fungus on host tissues, and they were related to drought stress. *c 2002 Elsevier Science Ltd. All rights reserved.

Keywords: charcoal rot; drought stress; histology; Phaseolus vulgaris L.; physiology; vegetative growth.

INTRODUCTION Water potentials from À12to À15 bars have been shown to increase predisposition to root rot pathogens [20]. Macrophomina phaseolina is an imperfect fungus which It has been suggested [21] that common bean cultivars causes charcoal rot in common bean #Phaseolus vulgaris with high leaf water potential under drought stress could L.), mainly under the dry and hot conditions of arid be resistant to root rot pathogens and inversely that regions of Mexico and other tropical and sub-tropical common bean germplasm resistant to charcoal rot could areas [1]. Drought stress may occur in any developmental be resistant to drought stress. Con®rmation of this stage of common bean [7] and increases crop pre- association would be useful for the identi®cation of disposition to infection by facultative parasites such as germplasm resistant to charcoal rot and/or drought stress. M. phaseolina, due to negative e€ects on host physiology. Reduced humidity in soils favours M. phaseolina develop- Drought stress reduces transpiration rate and stomatal ment in common bean under controlled [17]or®eld[8] conductance, water potential and its components osmotic conditions. Damage caused by charcoal rot is mainly and turgor potentials. In addition, water de®cit increases signi®cant in the early stages of common bean develop- carbohydrate content, which is available to the pathogen. ment. Physiological and histopathological alterations caused by M. phaseolina in common beans are poorly * To whom all correspondence should be addressed. E-mail: understood. In previous work, we studied the e€ect of [email protected] Abbreviations used in text: PWP, permanent wilting point; high temperatures/drought stress on charcoal rot devel- PDA, potato-dextrose-agar; VPD, vapour pressure de®cit; opment in two common bean cultivars. Charcoal rot did ANOVA, analysis of variance not a€ect transpiration rate and stomatal conductance, 0885-5765/02/$ - see front matter *c 2002 Elsevier Science Ltd. All rights reserved. 186 N. Mayek-PeÂrez et al. water, osmotic and turgor potentials. In addition, only Infestation levels drought stress increased charcoal rot development [17]. Two levels of infestation of M. phaseolina were evaluated: Histopathological alterations caused by M. phaseolina inoculated and uninoculated #control). Soil was infested during its infective process have not been characterized in using a colonized substrate #5 % w/w, 3000 + 250 common bean. In soybean #Glycine max), the fungus has cfu gÀ1 of dry substrate) of a highly virulent isolate of been shown to infect cotyledons, roots or stems in either M. phaseolina obtained from common bean plants pre-emergence or post-emergence stages. Microsclerotia collected in Cotaxtla, Veracruz, Mexico [16]. Stems of form appressoria over host epidermal cells, developing infected plants were sterilized with NaOCl 2.0 % and hyphae which enter between epidermal cells. The hyphae placed on Petri dishes containing potato-dextrose-agar grow inter- and intracellularly, and attack cells by #PDA). The plates were incubated at 308C for seven mechanical or enzymatic action. Intracellular coloniza- days. Inoculum was then prepared using rice seeds that tion occurs after lamella and cell wall disintegration [4]. were soaked overnight, rinsed three times in tap water Following epidermal and cortex invasion, M. phaseolina and placed in glass ¯asks. Seeds were sterilized at 1208C colonizes the vascular system developing microsclerotia for 2h during two consecutive days. Five 1 cm diameter on xylem vessels which may lead to their blockage. disks of PDA containing 8 day old M. phaseolina cultures Xylem vessel blocking causes wilt symptoms in soybean were placed in each ¯ask, and incubated at 308C for 15 and other hosts [12]. days. Colonized seeds were then dried at room tempera- Here, we report the characterization of the water ture for six days. Non-colonized substrate was applied in relations and histopathology of four common bean control pots at the same rate. cultivars during pathogenesis by M. phaseolina under drought stress in greenhouse conditions, in order to detect physiological or histological characteristics associated Experiment characteristics with resistance in common bean to M. phaseolina which could be useful criteria for the screening of bean Treatments were established using a completely random- germplasm for resistance to charcoal rot. ized design with ®ve replicates. The experimental unit was a plastic pot ®lled with 0.85 kg of methyl-bromide sterilized soil. The soil had a 9.0 % humidity at sowing and had been classi®ed as a sandy-clay soil with 15.4% MATERIALS AND METHODS #0.03 MPa) of ®eld capacity and 8.4% #0.15 MPa) of . Germplasm PWP, pH ˆ 6 7. Three seeds were sown in each pot. After emergence, one plant was mantained in each pot. Experiments were conducted under greenhouse con- Average maximum and minimum temperatures of 30.0/ ditions at Montecillo, Me xico #198210N, 988530W, and 19.28C were registered throughout the experiment. 2250 m above sea level). Four common bean cultivars were included, two resistant to M. phaseolina, BAT 477 and TLP 19, and two susceptible, Pinto UI-114 and Rio Water relations Tibagi [15, 21]. BAT 477, TLP 19, and Rio Tibagi À2 À1 Transpiration rate #g of H2Ocm s ), stomatal belong to Mesoamerican race, while Pinto UI-114 was resistance #s cmÀ1), and leaf temperature #8C) were classi®ed as race Durango [24]. BAT 477 and TLP 19 are determined using a portable porometer #Li-1600; Li-Cor improved lines from Colombia, and have growth habits Inc., Lincoln, NE, U.S.A.). Data were recorded in the III and II [23], respectively. Pinto UI-114 was developed ®ve replicates of each treatment at 20 #end of irrigation), in the U.S.A. and has growth habit type III, while Rio 28 #begining of PWP), 33 #®fth day after PWP), and 38 Tibagi is a landrace from Brazil with growth habit type #®fth day after recovery watering) days after sowing, from II. 12:00 to 13:00 h on each date of sampling. Porometer readings were taken on the central leaf from completely Humidity levels expanded trifoliate leaves exposed to solar radiation. Water potential #CW, bars) was measured in the same Two humidity levels were evaluated: irrigation and leaves used for porometer readings using a Scholander drought stress. Irrigated plants were cultivated under pump. After CW determinations, leaves were frozen in an available humidity level up 60 % throughout the liquid nitrogen #À1968C) to determine osmotic potential experiment. Drought stress consisted of the suspension of #C , bars) using a Wescor HR-33T #Wescor Inc., Logan, irrigation at 20 days after sowing, until plants reached the p U.S.A.) osmometer. Turgor potential #Cr, bars) was permanent wilting point #PWP). Plants were maintained calculated from the equation: under PWP for ®ve days, after which recovery watering was applied. Cr ˆ CW ‡ Cp Water relations, histopathology and growth of common bean 187 Vapour pressure de®cit #VPD, mbar) of each sample date petioles. Vegetative structures were dried in an oven at was calculated using the equation: 708C for ®ve days. The dry weight of each structure was then scored, and total dry weight and root/shoot ratios VPD ˆ es À e were calculated. where es, saturated vapour pressure at a given tempera- ture #millibars), e, saturated vapour pressure at actual Data analysis given temperature #millibars) Analysis of variance #ANOVA) was carried out for each During the ®fth day after PWP, relative water content variable. When ANOVA detected signi®cant di€erences #RWC, %) was determined. A completely expanded between treatments, means were separated using Tukey's single leaf was cut from a trifoliate leaf on each plant and values #P ˆ 0.05). Correlation coecients between dis- weighed #fresh weight, g). The leaf was then dissected ease severity ratings caused by M. phaseolina in irrigated and placed in distilled water for 2h. Leaves were dried and drought stressed plants and physiological and growth carefully to eliminate excessive water and weighed again characteristics were calculated. Statistical analysis was #maximum turgor weight, g). Each leaf was dried in an performed using SAS 98 for Windows #SAS Institute, oven at 708C for three days and then weighed #dry Cary, U.S.A.) software. weight, g). Relative water content was calculated using the equation:

RWC ˆ Fresh weight À Dry weight=Maximum turgor RESULTS weight À Dry weight†Â100 Water relations In order to determine drought stress e€ects on common bean physiology during charcoal rot development, Histopathology di€erent characterizations of water relations were carried Seedlings of BAT 477 and Pinto UI-114 subjected to all out. Drought stress reduced transpiration rate and humidity and/or M. phaseolina treatments were harvested increased stomatal resistance and leaf temperature in all at 28 and 33 days after sowing, and were washed in tap four common bean cultivars. Drought stress e€ects were water to separate soil particles. Each seedling was increased by inoculation of M. phaseolina. Transpiration dissected into leaves, hypocotyls, and roots. Roots and rate was lower in irrigated as compared to drought hypocotyls were cut in segments of 2cm length and ®xed stressed plants, while drought stressed plants showed the in FAA #50 Formaldehyde/5 ethanol/10 acetic acid/35 lowest stomatal resistances. Irrigated plants showed distilled water). Tissues were dehydrated using an variations in transpiration rate due to variations in ascending ethanol series #10 % to absolute), transferred VPD #28.0, 25.0, 28.2, and 31.2 mbars at 20, 28, 33, to xylene and were embedded in paran. Hypocotyl and 38 das, respectively). No di€erences were registered segments of 12 mm thickness were obtained using a throughout the sampling dates for transpiration rate, rotative microtome #Carl Zeiss, Germany) and placed on stomatal resistance or leaf temperature between resistant glass slides. Paran was removed from tissues using and susceptible common bean cultivars, under the same xylene and a descending ethanol series #96±50 %). treatment #Fig. 1), although BAT 477 showed the highest Tissues were re-hydrated using an ascending ethanol transpiration rate and the lowest stomatal resistances. series #50 % to absolute). Samples were stained with Drought stress reduced water and turgor potentials in safranin [5], and they were mounted on glass slides with all four common bean genotypes, while the osmotic synthetic resin. Tissues were observed with an optical potential was stable throughout samplings, humidity microscope #Carl Zeiss, Germany). levels or inoculation treatments. M. phaseolina increased the negative e€ects caused by drought stress on water and turgor potentials. Water and turgor potential variations Disease severity of charcoal rot and plant growth depended upon VPD variations, as did transpiration rate. Plants were harvested at 38 days after sowing and rinsed No di€erences were observed throughout sample dates for in tap water. The disease severity of M. phaseolina on roots water potential and its components among resistant or and hypocotyls was recorded in each experimental unit susceptible common bean cultivars, under similar con- using a scale with nine values, from 1 to 9, where 1 ˆ No ditions #Fig. 2), even though BAT 477 y TLP 19 showed visible symptoms to 9 ˆ More than 75 % of root or stem higher water and turgor potentials than Pinto UI-114 and tissues infected by the fungus [1]. We took values of 1±3 Rio Tibagi. as a resistance reaction, and 4±9 as a susceptible reaction. Relative water content was reduced by drought stress, Plants were dissected into leaves, roots and stems and and also slightly reduced by M. phaseolina. Under 188 N. Mayek-PeÂrez et al.

) 15 –1 s –2 (a) Ocm 2 10 I-C I-I D-C D-I

5

Transpiration rate (g of H 0 200 (b) ) –1 150

100

50 Stomatal resistance (s cm

0 40 (c)

35

30 Leaf temperature (°C) 25

20 20 28 33 38 20 28 33 38 20 28 33 38 20 28 33 38 Days after sowing

BAT 477 Pinto UI-114 Rio Tibagi TLP 19

FIG. 1. Variation of #a) transpiration rate, #b) stomatal resistance, #c) leaf temperature of four common bean cultivars grown under drought stress and M. phaseolina inoculation. #I ˆ Irrigated and D ˆ Drought stressed; In ˆ Inoculated and C ˆ Control). Vertical bars indicate Tukey's values among treatments #P ˆ 0.05) for each sampling date. Arrows indicate when recovery watering was applied to all treatments. irrigated conditions, relative water content was the same Charcoal rot disease severity and growth of common bean among cultivars, while drought stressed plants of M. phaseolina delayed emergence of common beans cultivars BAT 477 and TLP 19 showed the highest and disease severity ratings were increased under relative water content #Table 1). Water relations, histopathology and growth of common bean 189

5

(a)

0

–5

Water potential (bars) –10

I-C I-I D-C D-I

–15 5

(b)

0

–5

–10 Osmotic potential (bars)

–15

(c) 12

8

Turgor potential (bars) 4

0 20 28 33 38 20 28 33 38 20 28 33 38 20 28 33 38 Days after sowing

BAT 477 Pinto UI-114 Rio Tibagi TLP 19

FIG. 2. Variation of #a) water potential, #b) osmotic potential, #c) turgor potential of four common bean cultivars cultivated under drought stress and M. phaseolina inoculation. #I ˆ Irrigated and D ˆ Drought stressed; In ˆ Inoculated and C ˆ Control). Vertical bars indicate Tukey's values among treatments #P ˆ 0.05) for each sampling date. Arrows indicate when recovery watering was applied to all treatments. drought stress. Under irrigation, TLP 19 showed ing charcoal rot on susceptible common bean complete emergence later, while BAT 477 under cultivars Rio Tibagi and Pinto UI-114, while TLP stress conditions had the lowest days to emergence 19 under drought stress was susceptible to charcoal rot average. Arti®cial inoculation was ecient in develop- #Table 1). 190 N. Mayek-PeÂrez et al.

TABLE 1. Emergence, disease severity ratings, and relative water contents for four common bean cultivars grown under drought stress and M. phaseolina inoculation

Irrigation Drought stress

Relative water Relative water Factor Emergence Disease severity content Emergence Disease severity content

Inoculated plants d%d% BAT 477 11 4.089.4144.859.1 TLP 19 13 5.487.5127.455.7 Rio Tibagi 126 .287 .7128.852.1 Pinto UI-114 11 7.089.4128.050.0 Mean 125 .788.5137.354.2 Control plants BAT 477 8 1.089.98 1.064.3 TLP 19 8 1.089.58 1.061.1 Rio Tibagi 8 1.089.08 1.056.4 Pinto UI-114 8 1.088.88 1.055.8 Mean 8 1.089.38 1.059.4 Tukey #P ˆ 0.05) 1 3.026.01 3.026.0

Drought stress and charcoal rot decreased leaf area stress increased r coecients between charcoal rot and root, shoot and total dry weight of common beans. development and all other characteristics #Table 3). The drought stress/charcoal rot combination caused the highest reductions in all growth characteristics. Histopathology Pinto UI-114 and BAT 477 showed greater leaf area and dry mass accumulation than TLP 19 and Rio Infected plants showed the typical symptoms caused by Tibagi, under drought stress and/or M. phaseolina inocu- charcoal rot, such as deep and irregular necrotic lesions, lation #Table 2). that were extended across hypocotyl and root surfaces. Disease severity of M. phaseolina was negatively M. phaseolina infection patterns were similar in either correlated with transpiration rate, water, osmotic and resistant or susceptible genotypes. The fungus adhered to turgor potentials and relative water content, while disease the seed or cotyledon coats and then produced the severity showed a positive relationship with stomatal infective hyphae, infecting roots, cotyledons and/or resistance. No correlations were observed between char- hypocotyls. Charcoal rot development was faster in coal rot development and leaf temperature. Drought Pinto UI-114 than BAT 477, and under water stress as

TABLE 2. Shoot, root and total biomass, and root/shoot ratios for four common bean cultivars under drought stress and M. phaseolina inoculation

Irrigation Drought stress

Factor Leaf area Shoot dry Root dry Total dry Root/shoot Leaf area Shoot dry Root dry Total dry Root/shoot weight weight weight ratio weight weight weight ratio cm2 ggg cm2 ggg

Inoculated plants BAT 477 62.70.33 0.120 .45 0.36 42.70.240.120 .36 0.50 TLP 19 36.70.240.13 0.37 0.54 42.40.250.11 0.36 0.44 Rio Tibagi 21.50.230.11 0.34 0.48 14.90.22 0.14 0.36 0.64 Pinto UI-114 108.80.620 .22 0.84 0.36 50.10.35 0.13 0.48 0.37 Mean 57.40.36 0.15 0.50 0.44 37.50.270.13 0.39 0.49 Control plants BAT 477 174.51.020 .51 1.53 0.50 100.00.66 0.44 1.10 0.67 TLP 19 152.50.75 0.34 1.07 0.45 87.70.55 0.260.81 0.47 Rio Tibagi 171.30.720 .33 1.05 0.46 44.00.37 0.22 0.59 0.60 Pinto UI-114 181.31.33 0.65 1.98 0.49 91.30.70 0.45 1.15 0.64 Mean 169.90.96 0.46 1.41 0.48 80.80.57 0.34 0.91 0.60 Tukey #P ˆ 0.05) 28.50.31 0.14 0.40 0.2028.50.31 0.14 0.40 0.20 Water relations, histopathology and growth of common bean 191

TABLE 3. Correlation coecients /r) between disease severity to loss of turgor in occlused cells. Drought stressed plants ratings in inoculated common bean plants and some physiological partially recovered their transpiration rate and stomatal and growth characteristics resistances, after the recovery watering. During the drought stress period, abscisic acid is synthetized to Characteristic Disease severity on inoculated plants promote stomatal closure. In addition, drought stress Irrigated Drought stressed e€ects were increased by inoculation of M. phaseolina. Charcoal rot has been shown to increase stomatal ÐrÐ resistance and reduce transpiration rates in Vigna . . Transpiration rate À0 48** À0 55** aconitifolia [19] and sorghum [22]. Leaf temperature was Stomatal resistance 0.50** 0.58** Leaf temperature 0.22NS 0.24NS not a€ected by either drought stress or M. phaseolina, Water potential À0.41* À0.57** meanwhile both factors a€ected leaf temperature in Osmotic potential À0.38* À0.50** V. aconitifolia [19]. Since the transpiration rate is partially Turgor potential À0.42* À0.45** responsible for temperature increases in both cases, it can . . Relative water content À0 29NS À0 33* be assumed that leaf temperature stability is an important Leaf area À0.45** À0.58** Shoot dry weight À0.50** À0.60** characteristic in common bean. Root dry weight À0.48** À0.59** Drought stress reduced water and turgor potentials in Total dry weight À0.49** À0.58** all common bean genotypes, and charcoal rot increased the negative e€ects caused by drought stress on water and NO = No signi®cance at the 0.05 probability level. turgor potentials. Resistant genotypes showed higher *,** = Signi®cance at the 0.05 and 0.01 probability levels, respectively. water and turgor potentials than susceptible genotypes. Variable responses to charcoal rot on host physiology have been reported. While no signi®cant e€ect due to compared to irrigated conditions. Susceptible cultivars M. phaseolina on common bean physiology under drought showed typical symptoms after emergence, while BAT stress has been found [17], a negative relationship between 477 showed small lesions in the epidermal and external charcoal rot development and water potential in sorghum cortex cells only at the end of the study. has been detected [9]. In sorghum resistance to Microsclerotia in contact with the common bean M. phaseolina depends on the maintenance of physiological surface developed hyphae which penetrate between vigour produced by stable transpiration rates, that epidermal cells [Figs 3#b) and #c)], and then the fungus support an optimum carbohydrate and nutrient supply grew intercellularly [Fig. 3#d)]. Afterwards the fungus to the host [10]. Low water potentials in infected bean caused cellular collapse, necrosis of epidermal and cortex plants are caused by either changes in transpiration rate or cells in roots and hypocotyls of common bean [Figs 3#e)± absorption and water transport resistances. Stomatal #g)], and tissue disintegration which accelerated the resistance is increased when relative water contents falls death of the host [Figs 4#a)±#c)]. Parenchyma cells to 70 %, mainly due to stomatal closure and reduction in showed hypertrophy and hyperplasia [Figs 4#e) and water potentials. Since M. phaseolina colonizes epidermal, #f)] between the vascular cylinder and the cortex of cortical, and vascular cells, it develops microsclerotia hypocotyls, following disintegration of zones on the cortex which can block xylem vessels. These e€ects provoke wilt [Fig. 4#b)]. Damaged cells showed deposition of poly- symptoms in the host [12]. Results suggest the close phenols [Fig. 4#e)], while severe infections showed a association between high water potentials and resistance disintegration of the vascular system and signi®cant to charcoal rot in common beans, as occurs in sorghum disorganization of pith parenchyma [Fig. 4#c)]. While [9, 10]. Measurement of water potential during charcoal BAT 477 showed cellular damage in epidermis and cortex rot infection could be a useful parameter for screening parenchyma, Pinto UI-114 showed damage in epidermis, common bean germplasm resistant to charcoal rot. cortex, vascular cylinder, and pith. The disintegration of Turgor potential did not reach zero values during xylem and phloem parenchyma and pith cells in Pinto permanent wilting point in drought stressed plants, due to UI-114 was also detected. After colonization of epidermal the fact that the osmotic potential was reduced more than and cortical cells, the fungus colonized vascular cambium the water potential after the active accumulation of solutes and phloem [Figs 4#c) and #f)]. that maintain partial turgor of tissues. This mechanism is called osmotic adjustment [18] and was higher in BAT 477 than other cultivars. Osmotic adjustment could DISCUSSION have a signi®cant role in common bean resistance to Drought stress showed a signi®cant negative e€ect on M. phaseolina under drought stress, since it supports vigour transpiration rate and increased stomatal resistance in and physiological activity of the host. common bean. Transpiration rate was reduced by Relative water content was reduced by drought stress, increasing stomatal resistance, since stomata close due and also slightly reduced by M. phaseolina. M. phaseolina 192 N. Mayek-PeÂrez et al.

FIG. 3. Transverse sections of common bean cv. Pinto UI-114 hypocotyls, subjected to drought stress and M. phaseolina inoculation. #a) Healthy tissues, #b) and #c) a microsclerotium initiating pathogenesis, #d) cellular necrosis of epidermis, #e) cellular collapse and necrosis of cortex parenchyma, #f) necrosis of cortex, #g) collapse, necrosis and cellular disintegration in cortex and pith. has been shown to reduce relative water content of Drought stress and charcoal rot decreased growth and sorghum [22]. Sorghum cultivars resistant to charcoal rot dry matter accumulation patterns of common beans. The showed high relative water contents and lower reductions drought stress/charcoal rot combination caused the in the variation between irrigated and drought stressed highest reductions in all growth characteristics. Drought conditions as compared to susceptible cultivars. Charcoal stress reduces the vegetative growth of common beans, rot undergoes rapid development under strong water due its negative e€ect on cell division and expansion content depletion [22], therefore, cultivars that show rates [2]. In addition to the negative e€ects on reduced water depletion rates and a stable cellular turgor growth induced by drought stress, dry mass accumu- such as BAT 477, are resistant to charcoal rot. lation was reduced due to increased pre-emergence and Water relations, histopathology and growth of common bean 193

FIG. 4. Transverse sections of common bean cv. Pinto UI-114 hypocotyls, subjected to water stress and M. phaseolina inoculation. #a) cellular disintegration of cortex, #b) and #c) cortex, vascular cylinder and pith disintegration, #d) healthy pith tissues, #e) and #f) hypertrophy and hyperplasia of pith parenchyma. post-emergence seedling death percentages on inocu- cients between charcoal rot development and other lation of M. phaseolina. However, negative e€ects on characteristics. Results indicated that drought stress common bean growth by drought stress were greater increases the negative e€ects caused M. phaseolina in than those caused by charcoal rot [17]. common beans. Therefore, characterization and screen- Disease severity of M. phaseolina was negatively ing of common bean germplasm resistant to charcoal correlated with physiological parameters of all common rot will be most ecient under drought stress con- bean cultivars, and drought stress increased r coe- ditions. 194 N. Mayek-PeÂrez et al. Charcoal rot development was faster in Pinto UI-114 J. J. Vargas-Herna ndez, L. Iracheta-Donjuan, G. than BAT 477, and under water stress as compared to Valdovinos-Ponce, A. Gutie rrez-Larraza bal, R. Romer- irrigated conditions. Susceptible cultivars showed typical o-Co rdova, A. Galicia-Jua rez #Colegio de Postgradua- symptoms following emergence, while BAT 477 showed dos), R. Rico-Martõ nez and E. Pe rez-Molphe Balch small lesions in the epidermal and external cortex cells #Universidad Auto noma de Aguascalientes) for technical only at the end of the study. These symptoms have been support found in other hosts such as soybean, where M. phaseolina infects cotyledons due to adherence of microsclerotia to the seed coat during germination and emergence [6]. As demonstrated in this work, charcoal rot causes deep and REFERENCES irregular necrotic lesions that extend to hypocotyls and root surfaces in soybean [3], chickpea [26] and sorghum 1. 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Evaluacio n de genotipos de frijol e in¯uencia chickpea [27], garlic [11], maize [25], and guava [28]. de la temperatura con relacio n a la pudricio n carbonosa. M. phaseolina was capable of infecting the leaf epidermal Agricultura TeÂcnica en MeÂxico 18: 3±10. cells of Hydrilla verticillata, since these cells have an 8. DõÂaz-Franco A, Cortinas-Escobar H. 1988. E€ect of osmophilic layer in their walls [13]. Characterization of irrigation regimes on susceptibility of bean to Macropho- mina phaseolina. Journal of the Rio Grande Valley Horticultural the chemical nature of the osmophilic layer could help to Society 41: 47±50. determine the biochemical and anatomical basis of 9. Diourte M, Starr JL, Jeger MJ, Stack JP, Rosenow DT. resistance to charcoal rot in H. verticillata, which could 1995. Charcoal rot #Macrophomina phaseolina) resistance be applied to other hosts. and e€ects of water stress on disease development in BAT 477 showed resistance to charcoal rot under sorghum. 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The results sclerotia in soybean infected with Macrophomina phaseolina. presented here and further characterizations will provide Phytopathology 64: 156±157. useful information for designing an ecient breeding 13. Joye GF, Paul RN. 1992. Histology of infection of Hydrilla verticillata by Macrophomina phaseolina. Weed Science 40: strategy to produce common bean cultivars resistant 288±295. to charcoal rot in Mexico, based on physiological and 14. Karunakar RI, Kunwar IK, Satyaprasad K, Ramarao P. histopathological screening criteria. 1992. Histopathology of sorghum seedling roots infected by Macrophomina phaseolina. Phytophylactica 24: 325±327. 15. Mayek-PeÂrez N, Acosta-Gallegos JA, LoÂpez-CastanÄeda C, LoÂpez-Salinas E, CumpeaÂn-GutieÂrrez J, Acosta- We thank the Consejo Nacional de Ciencia y Tecnologõ a DõÂaz E. 1997. Resistance to Macrophomina phaseolina in #CONACyT), projects 3230P-B and J33785-B, for ®nan- common beans under ®eld conditions. Bean Improvement cial support. 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