An Anatomical and Physiological Basis for Flood-Mediated Rice Blast Field Resistance

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1 PEST MANAGEMENT: DISEASES An Anatomical and Physiological Basis for Flood-Mediated Rice Blast Field Resistance M.P. Singh, P.A. Counce, and F.N. Lee ABSTRACT Rice blast, a production-limiting rice pathogen in Arkansas and the world, is much more severe when susceptible varieties are grown in upland conditions as compared to continuous flood irrigation. This induced field partial resistance appears linked with concurrent anatomical and physiological changes during formation of aerenchyma tissue, under ethylene regulation, to facilitate root aeration as flooded rice plants adapt to anaerobic conditions. Increased ethylene within flooded plants has been linked to signaled plant defense responses which confer rice blast field resistance. To further understand the nature of flood induced resistance, we conducted a comparative anatomical study of leaf tissue of plants growing upland, flooded, or upland-treated with 50 ppm ethephon, which releases ethylene. Vascular leaf bundles from plants growing upland were poorly organized compared to those of continuous flooded plants with well organized enlarged cells within the leaf veins. From initial leaf infection sites on susceptible cultivars growing upland, the blast fungus completely colonized all cells and typically progressed within and laterally across the major and minor parallel leaf veins while completely colonizing cells of susceptible cultivars growing upland. With continuously flooded and upland-ethephon treated plants restricted the rate of fungal mycelium growth within and between the better organized major and minor lateral veins was reduced and thus overall lesion development limited. This restriction of the invasive fungus appeared to be structural and physiological in nature. With induced disease resistance, plant gene expression was altered in response to changing environment and hormone treatment. In nature there are multiple inducible defense mechanisms, such as rice blast resistance, which are controlled with a mix 217

2 AAES Research Series 517 of biotic and abiotic stress generated signals. The ability to effectively utilize these inducible defense systems depends entirely upon our ability to understand their natural mechanism of expression as well as the specificity and compatibility of regulating signaling systems. This research will aid in understanding and subsequent utilization of these processes. INTRODUCTION Rice blast, incited by Magnaporthe grisea, is less severe when plants are grown in continuous flood irrigation than when grown in an intermittent flood or an upland condition (Lee and McMinn, 1996). Kim et al. (1986) found flooding reduced the number of successful blast infections, the rate of lesion expansion and resulted in flattened disease gradients for blast disease development. Lesion number, conidia per lesion, and lesion size were significantly increased under upland conditions. Although controlled by minor genes, field-induced resistance interactions are nonspecific and appear to be more likely associated with plant physiological changes rather than changes within the plant canopy microclimate or virulence of the pathogen. Rice, a semi-aquatic plant, adapts to flood conditions through the formation of aerenchyma tissue which facilitates aeration of submerged roots. Involving cell-wall hydrolysis and cell lysis, aerenchyma cell development in flooded rice plants is regulated by ethylene and provides an excellent example of programmed cell death as described by Drew et al. (2000). Our research indicates that oxygen-limited conditions (hypoxia) in the flooded root zone signals the plant defense mechanisms conferring rice blast field resistance, likely through increased ethylene production (Singh et al., 2004). In that process, plant gene expression is altered in response to the changing environmental conditions. In nature these inducible defense mechanisms, such as rice blast resistance, are controlled with a mix of biotic and abiotic stress generated signals. Our ability to effectively utilize these inducible defense systems in an agricultural practice depends entirely upon our ability to understand their natural mechanism of expression as well as the specificity and compatibility of the signaling systems regulating their expression. This research will determine the physiological role of flooding in regulating disease development in rice. PROCEDURE The very susceptible M201, susceptible Cypress, and resistant Katy cultivars were grown in Dewitt silt loam soil in plastic pots 5 in. by 6 in. (H x W) with four 0.25-in. drainage holes. Each pot was planted with a ten to twelve seed/hill of test cultivars equidistantly spaced around the perimeter. Plants were grown for 4 weeks (V4), under simulated upland conditions by watering daily with deionized water and fertilized with with a 1% 20:20:20 N:P:K Peters solution one day prior to applying flood or a 50 ppm foliar-drench application of ethephon. For the flood 218

3 B.R. Wells Rice Research Studies 2003 condition, pots of upland plants were positioned inside larger pots, 9 in. by 14 in. (H x W), which were maintained near overflowing with deionized water to simulate plants growing in a 6-in. continuous flood. Greenhouse temperatures fluctuated between 27 C during the day and 24 C at night; RH was near 70%; and light photoperiod was 14 h day/10 h dark. Pathogenicity tests were conducted using M. grisea races IB-1, IB-49, IC-17, and IG-1 which commonly occur in Arkansas rice production fields. Spore suspensions, as a bulk of all races at 2 x 10 5 spores per ml, were prepared by dislodging spores from plates growing fungal cultures using distilled water containing 0.025% xanthan gum (Spectrum Inc., Calif.). Inoculum was applied to plants with an artist airbrush until runoff occurred. Immediately following inoculation, plants were moved into a dew chamber at 20 C and 100% RH in dark for 18 hours then returned to their original test positions. For whole leaf staining, the inoculated fifth leaf of test plants was removed then fixed in lactophenol (equal parts of phenol, lactic acid, glycerol, and distilled water) and completely decolorized by heating several times in a boiling water bath. Fixed leaf specimens were then placed in a solution of 0.75% NaCl acidified with 15% acetic acid and incubated at 95 C for 2 hours, dehydrated 5 minutes in a series of increasing alcohol concentrations of 30%, 50%, 70%, and 95% with a final 10 minute dehydration in absolute ethanol. Staining was accomplished by autoclaving the leaf tissue in a 0.05% aniline blue in lactophenol solution (equal parts of phenol, lactic acid, and absolute ethanol) at 121 C, 1 atm. for 15 min. After cooling, specimens were held one day in a differentiation mixture (equal parts of phenol, lactic acid, and absolute ethanol) to remove excess stain and then placed in anhydrous glycerol for histological examination. Leaf samples were stored in 70% ethanol until transversely hand sectioned with a razorblade. Selected fine sections were transferred to 90%, 95%, and absolute alcohol solutions for dehydration then mounted as with whole leaves. Microscopic examination was carried out using a 1950s Olympus BH series light microscope equipped with a Nikon Coolpix 950 digital camera attached with a CDCLA connector. RESULTS AND DISCUSSION The rice plant has a parallel venation arrangement (Fig. 1A) where leaf veins are continuous with those in the sheath and are connected of the stem through the nodes. On either side of the single centrally located leaf midrib there are four or five parallel main (large) veins which are separated by an additional four or five minor parallel veins (vascular bundles). The number of veins changes with leaf position but the distance between them remains unaltered. Large and small parallel vascular veins are interconnected by perpendicular transverse veins (Fig. 1A). The transverse and minor veins distribute water, nutrients, and photosynthetic products between leaf cells and main veins. Both large and small vascular bundles are surrounded by a layer of bundle sheath cells which transport nutrients and sugars and also serve as a temporary stor- 219

4 AAES Research Series 517 age vessels. Sometimes an additional layer of mestome sheath cells are present inside the bundle sheath cells. The xylem and phloem cells are situated within the vascular bundles which are filled with three to five layers of densely packed undifferentiated mesophyl cells. Epidermal cells with frequent stomata are arranged in rows alongside the veins. Large motor (bulliform) cells are located between stomatal rows on the upper (adaxial) side of the leaf expand and contract to regulate stomata opening and leaf rolling. The blast fungus spore typically germinates in free water, forcefully penetrates the waxy layer and cell walls of the leaf surface, invades the affected cells and then grows between and within tissue, with visible symptoms exhibited within three to five days. Various growth factors, nutrients, and environmental conditions impact the rate of lesion development. Whole leaf mounts show blast lesions as observed on plants growing uplanduntreated, flooded-untreated or upland-ethephon treated (Singh et.al., 2004). With upland M-201 and Cypress, spindle-shaped blast lesions resulted from rapid colonization along the infected veins and through transverse veins to adjacent lateral veins, (Fig. 2A,B). Parallel and perpendicular lesion development was restricted with untreatedflood and upland-ethephon treated plants. Katy shows only necrotic flecks at infection sites with all treatments. Response to treatment was evident when transverse anatomical sections were observed under the light microscope (Fig. 1). In contrast to the more loosely arranged veins in tissue from upland plants, veins of flooded plants exhibited a highly developed aerenchyma in main midrib with enlarged major and minor veins and well developed transverse veins (Fig. 1A). The large leaf veins of flooded and ethephontreated plants consisted of a clearly defined arrangement of the bundle sheath, xylem phloem, and supporting fiber with lysigenous aerenchyma tissue being formed between vascular bundles and connected with the stomata. Blast-fungus infected cells of susceptible plants appeared light brown or grayish with stained cell walls while hypersensitive and necrotic cells of resistant plants were filled with dark cytoplasmic granulations (Fig. 1 and 2). With very susceptible lesions of M-201 and upland Cypress, the upper epidermis, mesophyll, vascular bundles, bundle sheath, bulliforms cells, and even lower epidermis were colonized by the fungus. In advanced infections, fungal hyphae were present in all affected cells of upland leaf tissue with profuse external growth and sporulation. In flooded and ethephon-treated plants, fungal colonization of tissue between vascular bundles extended upward to epidermal layers but linear and lateral growth appears constrained by the enlarged bundle sheaths and by the transverse veins (Fig. 1B). These restrictions effectively restricted overall lesion development. We presume the reduced colonization of flood- and ethephon-treated plants was due to physical and physiological barriers established by the enlarged and extended bundle sheaths, the transverse veins and associated air spaces which limited the supply of water and nutrients to the fungus. In that process, plant gene expression was altered in response to changing environmental conditions. In nature, these inducible 220

5 B.R. Wells Rice Research Studies 2003 defense mechanisms, as with rice blast field resistance, are controlled with a mix of biotic and abiotic stress generated signals. Our ability to effectively utilize these inducible defense systems in an agricultural practice depends entirely upon our ability to understand and manipulate their natural mechanism of expression as well as the specificity and compatibility of the signaling systems regulating their expression. This research advances that effort. SIGNIFICANCE OF FINDINGS The research was conducted because the mechanism of flood-induced blast field resistance is poorly understood. Our observations provide a structural rationalization in conjunction with obvious physiological or metabolic components for expression of partial resistance. This information will be of value in future research designed to manipulate and optimize this induced response while developing high yielding blast field resistant varieties and/or practical control recommendations for growing very high-yielding blast-susceptible cultivars such as Wells and Francis. LITERATURE CITED Drew, M.C., C.J. He, and P.W. Morgan Programmed cell death and aerenchyma formation in roots. Trends Plant Sci. 5(3): Kim, C.H., M.C. Rush, and D.R. MacKenzie Flood-mediated resistance to the rice blast disease. Pages in: The Wetlands and Rice in Subsaharan Africa. A.S.R. Juo and J.A. Lowe, eds. International Institute of Tropical Agriculture, Ibadan. Lee, F.N. and T.A. McMinn Preliminary report on continuous flood depth influence on blast (Pyricularia grisea) in rice. In: R.J. Norman and B.R. Wells (eds.). Arkansas Rice Research Studies University of Arkansas Agricultural Experiment Station Research Series 453: Fayetteville, Ark. Singh, M.P. F.N. Lee, P.A. Counce, and J.H. Gibbons Mediation of partial resistance to rice blast through anaerobic induction of ethylene. Phytopathology 94:

AAES Research Series 517 Fig. 1. Characteristics of the Cypress cultivar when grown either upland, flooded, or upland-treated with 50 ppm ethephon.

6 AAES Research Series 517 Fig. 1. Characteristics of the Cypress cultivar when grown either upland, flooded, or upland-treated with 50 ppm ethephon. A. Characteristic vein structure in disease-free leaves. B. Relative fungal growth and lateral lesion development seven-days post inoculation. 222

7 B.R. Wells Rice Research Studies 2003 Fig. 2. Seven-day post inoculation rice blast lesions on rice cultivars M-201, Cypress, and Katy. A. Typical lesions observed when grown either upland, flooded, or upland-treated with 50 ppm ethephon. B. Relative surface sporulation and lesion development at the different treatments. 223

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23 4 Leaves Slide 1 of 32 23 4 Leaves 1 of 32 Leaf Structure The structure of a leaf is optimized for absorbing light and carrying out photosynthesis. 2 of 32 Leaf Structure To collect sunlight, most leaves have thin, flattened