- Email: [email protected]
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor
Physiological and Molecular Plant Pathology (2002) 61, 267±271 doi:10.1006/pmpp.2003.0440
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor M A R I A E U G E N I A A G U E RO* , A M A N D A G E V E N S and R A L P H L . N I C H O L S O N{ Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155, U.S.A. (Accepted for publication 30 September 2002) The concentration of 3-deoxyanthocyanidins from inoculated sorghum exhibited a sigmoid curve for accumulation between 10 and 32 h after inoculation. Luteolinidin and apigeninidin were the most abundant deoxyanthocyanidins present. Methoxy-apigeninidin and the caeic acid ester of arabinosyl 5-O-apigeninidin were detected but their concentrations were very low. At the time of inoculation, conidia of Cochliobolus heterostrophus were brown and cells were turgid. Host tissue was colorless and no symptoms were evident. Spectrophotometry and high performance liquid chromatography (HPLC) analyses showed that the 3-deoxyanthocyanidin phytoalexins were not present at the time of inoculation. Spectrophotometry did not detect deoxyanthocyanidins in plant extracts until 10±12 h post inoculation. In contrast, HPLC analysis showed that inoculated tissue released small amounts of luteolinidin and apigeninidin by 4 h after inoculation. It is the speed of this response that is unusual. After inoculation, both host cells and conidia appeared red. Phytoalexin-containing inclusions were released from the host tissue and became associated with the extracellular sheath of the dead conidia. c 2003 Elsevier Science Ltd. All rights reserved. * Keywords: Cochliobolus heterostrophus; phytoalexin; Sorghum bicolor; spectrophotometry.
INTRODUCTION As a mechanism of defence against pathogen attack or other stress, some plants produce antimicrobial compounds called phytoalexins. More than 300 phytoalexins have been identi®ed and characterized. Phytoalexins are typically secondary metabolites [1, 7, 9]. Sorghum (Sorghum bicolor L.) produces 3-deoxyanthocyanidin phytoalexins in a site-speci®c response to attempted fungal infection during both resistant non-host and host-speci®c resistant responses [5, 6]. These pigmented compounds are a class of low molecular weight ¯avonoids that accumulate in infected tissue at higher concentrations than required for fungitoxicity [6, 10]. Since the demonstration that phytoalexins serve as resistance factors in the defence of plants against microorganisms [4], many investigators have questioned when, where and how these compounds are produced. The 3-deoxyanthocyanidins appear to be produced as sub cellular inclusions [10, 11] and at concentrations that exceed levels necessary for fungitoxicity [13]. * This study is dedicated to Ms. Maria Eugenia Aguero who died suddenly during the pursuit of her Doctoral Investigations { To whom correspondence should be addressed. E-mail: [email protected]
0885-5765/03/$ - see front matter
The deoxyanthocyanidin inclusions may be present as vesicles that are derivatives of the ER-Golgi apparatus [10, 11]. Expression of fungitoxicity by the phytoalexins occurs when the compounds are released from the inclusions [11]. In spite of our accumulated knowledge, we still do not understand how these compounds aect fungal growth and development. In the present study, we used light microscopy, spectrophotometry and high performance liquid chromatography (HPLC) analysis to analyse events that occur at dierent stages through 40 h post inoculation in a non-host-pathogen interaction. The major objective was to determine when the phytoalexins are released from plant tissue, the concentration of phytoalexins in the mixture released, and the composition of the complex of compounds. It was our intent to begin to understand how the 3-deoxyanthocyanidins aect fungal development.
MATERIALS AND METHODS
Plant material, fungal culture and inoculation Seed of sorghum (Sorghum bicolor L cv.DK18) were imbibed in water (12 h). Twenty-®ve seed were planted in rolls of germination paper and incubated in the dark for 4 d at 25+28C. Seedlings, with uniformly etiolated c 2003 Elsevier Science Ltd. All rights reserved. *
268
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mesocotyls ( ®rst internodes) were selected and inoculated with a conidial suspension of Cochliobolus heterostrophus (Drechs.) Drechs. perfect stage of Bipolaris maydis (Nisik. and Miy). This fungus was chosen because previous studies had shown that its attempted penetration elicits an extremely rapid phytoalexin response [6]. Fungal cultures were maintained on potato dextrose agar under constant illumination at 25+28C. For inoculation, conidial suspensions were prepared by ¯ooding the surface of 14 day old cultures with distilled water amended with Tween 20 (0.1 ml 100 ml 1 sterile distilled water) as a wetting agent and then gently scraping the surface with a spatula to dislodge conidia. The suspension was ®ltered through muslin cloth to remove hyphal fragments. The conidial suspension was adjusted to an approximate concentration of 106 conidia ml 1. The suspension was applied as a ®ne mist with an atomizer. To ensure that the suspension was uniformly applied, plants were inoculated and then the inoculum was allowed to settle onto the tissue for a period of 5 min. The plants were then inoculated again, incubated in the dark at 25+28C and placed in a black bag to ensure high RH (near 100 %) during the experiment. From 0 to 40 h post inoculation (hpi), samples from inoculated plants were taken at intervals of 2 h. For each sample, mesocotyls from 125 seedlings were excised and placed in a tube with 45 ml of HPLC grade methanol and maintained at 48C overnight. This process allowed a majority of phytoalexins to leach from the host tissue. Thirty milliliter of each methanol extract were ®ltered under vacuum through a Millipore nitrocellulose ®lter (0.8 mm pore diameter), and maintained at 48C until concentration and composition analyses were performed. The concentration of the 3-deoxyanthocyanidin mixtures of compounds was determined by spectrophotometry as described below. The composition of the extract was determined by HPLC as described previously [3]. The remaining 15 ml of the methanol extract were not ®ltered and were used to perform light microscopy as described later. The experiment was done in triplicate.
Spectrophotometric analyses and concentration of phytoalexins released from infected tissue Absorbance in the visual light range (280±600 nm) was measured for each sample. Quartz micro-cuvets were used and 500 ml of each sample were analysed. According to the Lambert-Beer Law (A C E L), the concentration (C) of the mixture of 3-deoxyanthocyanidins in the extract was determined as the division of absorbance (A) at 506 nm by the product of the molar extinction coecient (E) and length that the light traveled through the sample (L). Because the phytoalexins exist as a mixture of ®ve compounds the molar extinction coecient used was that of luteolinidin and 5-methoxyluteolinidin
(13 800 m 1 cm 1), the principal fungitoxic components of the phytoalexin complex [3].
Light microscopy Strips of epidermal tissue were removed from internodes that had been submerged in distilled water. The strips (5±10) were mounted on a slide in distilled water, examined with bright®eld and/or Nomarski interference optics, and imaged with a Nikon FX-35DX camera, using Kodak Ektachrome 64 K tungsten color ®lm.
RESULTS The concentration of 3-deoxyanthocyanidins extracted into methanol from inoculated tissue is shown by a sigmoid curve that explains 84 % of the total variation observed (Fig. 1). The curve exhibited a high rate of accumulation between 10 and 32 hpi and low rates of accumulation at initial and ®nal stages (Fig. 1). Luteolinidin and apigeninidin were the most abundant deoxyanthocyanidins present during the period studied (Fig. 2). Methoxy-apigeninidin and the caeic acid ester of arabinosyl 5-O-apigeninidin were also detected by HLPC, but their relative concentration in the mixture was always lower than that of the other compounds (Fig. 2). The 5-methyl ether of luteolinidin was not detected. At the time of inoculation, conidia were brown; their cells were turgid and the internal cellular structure was easily distinguished. The host tissue was colorless and no symptoms were evident at this time. At 2 hpi conidia looked as those that had been observed at 0 hpi and many were starting to germinate. Spectrophotometry and HPLC analyses also showed that the 3-deoxyanthocyanidin phytoalexins were not present at early stages post inoculation (Figs 1 and 2). At 4 hpi conidia were brown and many appeared to be attempting to penetrate into the host. Even when host tissue was white, faintly yellow, small, round to isodiametric inclusions were observed in the tissue epidermis [Figs 3(a) and (c)]. Nomarski optics also revealed similar spheres on the upper surfaces of some conidia (Fig. 3(c)), suggesting that inclusions had been released from the host tissue. At 4 and 6 hpi, these yellow inclusions were seen more frequently than the red inclusions observed at later times after inoculation [Figs 3(d)±( f)]). At this time, when spectrophotometry did not detect any deoxyanthocyanidins in the plant extract (Fig. 1), HPLC showed that infected, etiolated mesocotyls released small amounts of luteolinidin and apigeninidin (Fig. 2). Similar results were obtained at 6 and 8 hpi, but the relative concentration of apigeninidin rose signi®cantly (Fig. 2).
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor
269
F I G . 1. Spectrophotometric analysis of the complex of 3-deoxyanthocyanidin compounds that accumulated in etiolated mesocotyls of sorghum cv. DK18 from 0 to 40 h post inoculation. Absorbance was measured at 506 nm. Phytoalexin concentration is expressed in mM. Each point is the average of three replications and the curve re¯ects the change in concentration over time.
F I G . 2. Relative A480 due to each of the 3-deoxyanthocyanidin compounds revealed by HPLC analysis. Analysis of extracts from etiolated mesocotyls of sorghum cv. DK18 from 0 to 40 hpi. For each sampling time, the points indicate the percent of total A480 in the extract that was represented by each 3-deoxyanthocyanidin. The balance of A480 was due to unidenti®ed 480 nm-absorbing compounds. Points are the averages of three replications. L, luteolinidin; A, apigeninidin; MA, Methoxy-apigeninidin; CA, the caeic acid ester of arabinosyl 5-O-apigeninidin.
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M. E. Aguero et al.
F I G . 3. Development of Cochliobolus heterostrophus on sorghum mesocotyls and production of deoxyanthocyanidin phytoalexins by the host tissue. (a) Germinated conidium of C. heterostrophus at 4 h after inoculation, Nomarski optics. (b) Germinated conidium of C. heterostrophus at 6 h after inoculation. Note the underlying host cells are pigmented red due to accumulation of deoxyanthocyanidin phytoalexins. (c) Between 4 and 6 h many host cells have developed inclusions (arrow) that surround the tip of the conidium. Inclusions also line one edge of the conidium, and similar spheres are on the upper surface of the conidium. The inclusions shown here are not yet pigmented, Nomarski optics. (d) A high concentration of, red colored phytoalexin inclusions surround an ungerminated conidium at 9 h after inoculation. Figures (d), (e), and ( f) are of conidia in suspension. (e) and ( f) By 20±24 h numerous round, red phytoalexin inclusions have accumulated around both germinated and ungerminated conidia, respectively. (g) Germinated conidium on the surface of a mesocotyl. Note the intense pigmentation of the mesocotyl and collapse of the germ tube.
At 6 hpi both the host tissue and the conidia began to change color, and conidia appeared less healthy than those seen at earlier stages after inoculation. At 10 hpi,
a yellow color was evident in host tissue where germinated conidia had attempted to penetrate. Thus they looked like Fig. 3(a). Chemical identi®cation of the yellow material was not attempted yet the yellow color would seem to infer that the material might be ¯avonoid in nature. Consistent with this, an increase in luteolinidin content in the plant extract was also detected by HPLC (Fig. 2). At 16 hpi both the host tissue and the conidia appeared red and looked like Fig. 3(b). It was clear that the highest accumulation of phytoalexins occurred at sites where the fungus originally attempted to penetrate (Fig. 3(b)). Appearing at 9 hpi and very evident after 20 hpi, a high concentration of round, red colored phytoalexin inclusions was observed in samples and the majority of these inclusions had accumulated around conidia [Figs 3(d)±(g)]. At 24 hpi the intense red coloration in the host tissue and the pathogen was due to a high concentration of phytoalexins in inclusions and associated with conidia [Figs 3(d)±(g)]. The conidia appeared to have taken up the compounds resulting in conidial death as indicated by collapse and dehydration of germ tubes (Fig. 3(g)). At 38 and 40 hpi many phytoalexin inclusions surrounded dead conidia and both the inclusions and the conidia were of the same intense red color and looked like Figs 3(d)±(g). This observation coincided with high concentrations of luteolinidin and apigeninidin in plant extracts (Fig. 2). The accumulation of phytoalexin inclusions was typically higher at the ends of the conidia, the sites from which germination occurred [Figs 3(d) and (e)]. Observations of germinated conidia at 40 hpi showed that the highest concentrations of inclusions were around germ tubes, as in Fig. 3(e). It is known that substantial quantities of extracellular material are released as part of the fungal germination process [8, 12]. Based on these observations, we propose that phytoalexin inclusions are `trapped' by extracellular sheath material that conidia release during the germination process (Fig. 3(e)). It is possible that this is the site where the 3-deoxyanthocyanidin compounds initiate their toxic eects.
DISCUSSION Accumulation of 3-deoxyanthocyanidins showed the same behavior as that of previous work [2]. The contribution of this work to the understanding of this resistant response is that we detected, by HPLC analysis, that the 3-deoxyanthocyanidin compounds were released hours earlier than previously assumed [2]. Thus, we may infer that host tissue can rapidly detect the presence of the pathogen and as soon as the fungus starts to attempt penetration the host initiates its defences.
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor The presence of free inclusions in water indicates that these phytoalexins are not as water soluble as previously thought [11]. It is also possible that the inclusions are derived from the ER-Golgi complex and remain membrane-enclosed. The composition of phytoalexins in the inclusions may de®ne inclusion color. The release of these compounds seems to depend on time after inoculation (Fig. 2). At 10 hpi several inclusions were composed primarily of luteolinidin and apigeninidin (Fig. 2) and this could account for host tissue color. HPLC analyses allowed us to detect the 3-deoxyanthocyanidins earlier than expected. The sensitivity of HPLC analyses may explain the dierence between our detection of phenols from 0 to 8 hpi and earlier reports. Thus, accumulation of phytoalexins in the sorghumC. heterostrophus interaction occurs much more rapidly than previously thought. It is also clear that phytoalexin inclusions become associated with the extracellular mucilage produced by the pathogen during germination.
REFERENCES 1. Grayer RJ, Harborne JB. 1994. A survey of antifungal compounds from higher plants, 1982±1993. Phytochemistry 37: 19±42. 2. Hipskind JG, Hanau R, Leite B, Nicholson RL. 1990. Phytoalexin accumulation in sorghum: identi®cation of an apigenidin acyl ester. Physiological and Molecular Plant Pathology 36: 381±396. 3. Lo SC, de Verdier K, Nicholson RL. 1999. Accumulation of 3-deoxyanthocyanidin phytoalexins and resistance to Colletotrichum sublineolum in sorghum. Physiological and Molecular Plant Pathology 55: 263±273.
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4. MuÈller KO, Boerger H. 1940. Experimentelle Untersuchungen uÈber die Phytophthora Resistenz der Kartoel, Zugleich ein Beitrag zum Problem der Erwobenen Resistenz. Arbeiten aus der Biologischen Reichsantalt fuÈr Land und Fortwurtschaft 23: 189±231. 5. Nicholson RL, Jamil FF, Snyder BA, Lue WL, Hipskind J. 1988. Phytoalexin synthesis in the juvenile sorghum leaf. Physiological and Molecular Plant Pathology 33: 271±278. 6. Nicholson RL, Kollipara SS, Vincent JR, Lyons PC, Cadena-Gomez G. 1987. Phytoalexin synthesis by the sorghum mesocotyl in response to infection by pathogenic and non-pathogenic fungi. Proceedings of the National Academy of Sciences, USA 84: 5520±5524. 7. Nicholson RL, Wood KV. 2001. Phytoalexins and secondary products, where are they and how can we measure them?. Physiological and Molecular Plant Pathology 59: 63±69. 8. Nielsen KA, Nicholson RL, Carver TLW, Kunoh H, Oliver RP. 2000. First touch: An immediate response to surface recognition in conidia of Blumeria graminis. Physiological and Molecular Plant Pathology 56: 63±70. 9. Smith CJ. 1996. Accumulation of phytoalexins: defense mechanism and stimulus response system. New Phytologist 132: 1±45. 10. Snyder BA, Leite B, Hipskind J, Butler LG, Nicholson RL. 1991. Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site. Physiological and Molecular Plant Pathology 39: 463±470. 11. Snyder BA, Nicholson RL. 1990. Synthesis of phytoalexins in Sorghum as a site-speci®c response to fungal ingress. Science 248: 1585±1688. 12. Sugui JA, Leite B, Nicholson RL. 1998. Partial characterization of the extracellular matrix released onto hydrophobic surfaces by conidia and conidial germlings of Colletotrichum graminicola. Physiological and Molecular Plant Pathology 52: 411±425. 13. Wharton PS, Nicholson RL. 2000. Temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoalexins and the anthocyanin, cyaniding 3-dimalonyl glucoside. New Phytologist 145: 457±469.
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor M A R I A E U G E N I A A G U E RO* , A M A N D A G E V E N S and R A L P H L . N I C H O L S O N{ Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155, U.S.A. (Accepted for publication 30 September 2002) The concentration of 3-deoxyanthocyanidins from inoculated sorghum exhibited a sigmoid curve for accumulation between 10 and 32 h after inoculation. Luteolinidin and apigeninidin were the most abundant deoxyanthocyanidins present. Methoxy-apigeninidin and the caeic acid ester of arabinosyl 5-O-apigeninidin were detected but their concentrations were very low. At the time of inoculation, conidia of Cochliobolus heterostrophus were brown and cells were turgid. Host tissue was colorless and no symptoms were evident. Spectrophotometry and high performance liquid chromatography (HPLC) analyses showed that the 3-deoxyanthocyanidin phytoalexins were not present at the time of inoculation. Spectrophotometry did not detect deoxyanthocyanidins in plant extracts until 10±12 h post inoculation. In contrast, HPLC analysis showed that inoculated tissue released small amounts of luteolinidin and apigeninidin by 4 h after inoculation. It is the speed of this response that is unusual. After inoculation, both host cells and conidia appeared red. Phytoalexin-containing inclusions were released from the host tissue and became associated with the extracellular sheath of the dead conidia. c 2003 Elsevier Science Ltd. All rights reserved. * Keywords: Cochliobolus heterostrophus; phytoalexin; Sorghum bicolor; spectrophotometry.
INTRODUCTION As a mechanism of defence against pathogen attack or other stress, some plants produce antimicrobial compounds called phytoalexins. More than 300 phytoalexins have been identi®ed and characterized. Phytoalexins are typically secondary metabolites [1, 7, 9]. Sorghum (Sorghum bicolor L.) produces 3-deoxyanthocyanidin phytoalexins in a site-speci®c response to attempted fungal infection during both resistant non-host and host-speci®c resistant responses [5, 6]. These pigmented compounds are a class of low molecular weight ¯avonoids that accumulate in infected tissue at higher concentrations than required for fungitoxicity [6, 10]. Since the demonstration that phytoalexins serve as resistance factors in the defence of plants against microorganisms [4], many investigators have questioned when, where and how these compounds are produced. The 3-deoxyanthocyanidins appear to be produced as sub cellular inclusions [10, 11] and at concentrations that exceed levels necessary for fungitoxicity [13]. * This study is dedicated to Ms. Maria Eugenia Aguero who died suddenly during the pursuit of her Doctoral Investigations { To whom correspondence should be addressed. E-mail: [email protected]
0885-5765/03/$ - see front matter
The deoxyanthocyanidin inclusions may be present as vesicles that are derivatives of the ER-Golgi apparatus [10, 11]. Expression of fungitoxicity by the phytoalexins occurs when the compounds are released from the inclusions [11]. In spite of our accumulated knowledge, we still do not understand how these compounds aect fungal growth and development. In the present study, we used light microscopy, spectrophotometry and high performance liquid chromatography (HPLC) analysis to analyse events that occur at dierent stages through 40 h post inoculation in a non-host-pathogen interaction. The major objective was to determine when the phytoalexins are released from plant tissue, the concentration of phytoalexins in the mixture released, and the composition of the complex of compounds. It was our intent to begin to understand how the 3-deoxyanthocyanidins aect fungal development.
MATERIALS AND METHODS
Plant material, fungal culture and inoculation Seed of sorghum (Sorghum bicolor L cv.DK18) were imbibed in water (12 h). Twenty-®ve seed were planted in rolls of germination paper and incubated in the dark for 4 d at 25+28C. Seedlings, with uniformly etiolated c 2003 Elsevier Science Ltd. All rights reserved. *
268
M. E. Aguero et al.
mesocotyls ( ®rst internodes) were selected and inoculated with a conidial suspension of Cochliobolus heterostrophus (Drechs.) Drechs. perfect stage of Bipolaris maydis (Nisik. and Miy). This fungus was chosen because previous studies had shown that its attempted penetration elicits an extremely rapid phytoalexin response [6]. Fungal cultures were maintained on potato dextrose agar under constant illumination at 25+28C. For inoculation, conidial suspensions were prepared by ¯ooding the surface of 14 day old cultures with distilled water amended with Tween 20 (0.1 ml 100 ml 1 sterile distilled water) as a wetting agent and then gently scraping the surface with a spatula to dislodge conidia. The suspension was ®ltered through muslin cloth to remove hyphal fragments. The conidial suspension was adjusted to an approximate concentration of 106 conidia ml 1. The suspension was applied as a ®ne mist with an atomizer. To ensure that the suspension was uniformly applied, plants were inoculated and then the inoculum was allowed to settle onto the tissue for a period of 5 min. The plants were then inoculated again, incubated in the dark at 25+28C and placed in a black bag to ensure high RH (near 100 %) during the experiment. From 0 to 40 h post inoculation (hpi), samples from inoculated plants were taken at intervals of 2 h. For each sample, mesocotyls from 125 seedlings were excised and placed in a tube with 45 ml of HPLC grade methanol and maintained at 48C overnight. This process allowed a majority of phytoalexins to leach from the host tissue. Thirty milliliter of each methanol extract were ®ltered under vacuum through a Millipore nitrocellulose ®lter (0.8 mm pore diameter), and maintained at 48C until concentration and composition analyses were performed. The concentration of the 3-deoxyanthocyanidin mixtures of compounds was determined by spectrophotometry as described below. The composition of the extract was determined by HPLC as described previously [3]. The remaining 15 ml of the methanol extract were not ®ltered and were used to perform light microscopy as described later. The experiment was done in triplicate.
Spectrophotometric analyses and concentration of phytoalexins released from infected tissue Absorbance in the visual light range (280±600 nm) was measured for each sample. Quartz micro-cuvets were used and 500 ml of each sample were analysed. According to the Lambert-Beer Law (A C E L), the concentration (C) of the mixture of 3-deoxyanthocyanidins in the extract was determined as the division of absorbance (A) at 506 nm by the product of the molar extinction coecient (E) and length that the light traveled through the sample (L). Because the phytoalexins exist as a mixture of ®ve compounds the molar extinction coecient used was that of luteolinidin and 5-methoxyluteolinidin
(13 800 m 1 cm 1), the principal fungitoxic components of the phytoalexin complex [3].
Light microscopy Strips of epidermal tissue were removed from internodes that had been submerged in distilled water. The strips (5±10) were mounted on a slide in distilled water, examined with bright®eld and/or Nomarski interference optics, and imaged with a Nikon FX-35DX camera, using Kodak Ektachrome 64 K tungsten color ®lm.
RESULTS The concentration of 3-deoxyanthocyanidins extracted into methanol from inoculated tissue is shown by a sigmoid curve that explains 84 % of the total variation observed (Fig. 1). The curve exhibited a high rate of accumulation between 10 and 32 hpi and low rates of accumulation at initial and ®nal stages (Fig. 1). Luteolinidin and apigeninidin were the most abundant deoxyanthocyanidins present during the period studied (Fig. 2). Methoxy-apigeninidin and the caeic acid ester of arabinosyl 5-O-apigeninidin were also detected by HLPC, but their relative concentration in the mixture was always lower than that of the other compounds (Fig. 2). The 5-methyl ether of luteolinidin was not detected. At the time of inoculation, conidia were brown; their cells were turgid and the internal cellular structure was easily distinguished. The host tissue was colorless and no symptoms were evident at this time. At 2 hpi conidia looked as those that had been observed at 0 hpi and many were starting to germinate. Spectrophotometry and HPLC analyses also showed that the 3-deoxyanthocyanidin phytoalexins were not present at early stages post inoculation (Figs 1 and 2). At 4 hpi conidia were brown and many appeared to be attempting to penetrate into the host. Even when host tissue was white, faintly yellow, small, round to isodiametric inclusions were observed in the tissue epidermis [Figs 3(a) and (c)]. Nomarski optics also revealed similar spheres on the upper surfaces of some conidia (Fig. 3(c)), suggesting that inclusions had been released from the host tissue. At 4 and 6 hpi, these yellow inclusions were seen more frequently than the red inclusions observed at later times after inoculation [Figs 3(d)±( f)]). At this time, when spectrophotometry did not detect any deoxyanthocyanidins in the plant extract (Fig. 1), HPLC showed that infected, etiolated mesocotyls released small amounts of luteolinidin and apigeninidin (Fig. 2). Similar results were obtained at 6 and 8 hpi, but the relative concentration of apigeninidin rose signi®cantly (Fig. 2).
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor
269
F I G . 1. Spectrophotometric analysis of the complex of 3-deoxyanthocyanidin compounds that accumulated in etiolated mesocotyls of sorghum cv. DK18 from 0 to 40 h post inoculation. Absorbance was measured at 506 nm. Phytoalexin concentration is expressed in mM. Each point is the average of three replications and the curve re¯ects the change in concentration over time.
F I G . 2. Relative A480 due to each of the 3-deoxyanthocyanidin compounds revealed by HPLC analysis. Analysis of extracts from etiolated mesocotyls of sorghum cv. DK18 from 0 to 40 hpi. For each sampling time, the points indicate the percent of total A480 in the extract that was represented by each 3-deoxyanthocyanidin. The balance of A480 was due to unidenti®ed 480 nm-absorbing compounds. Points are the averages of three replications. L, luteolinidin; A, apigeninidin; MA, Methoxy-apigeninidin; CA, the caeic acid ester of arabinosyl 5-O-apigeninidin.
270
M. E. Aguero et al.
F I G . 3. Development of Cochliobolus heterostrophus on sorghum mesocotyls and production of deoxyanthocyanidin phytoalexins by the host tissue. (a) Germinated conidium of C. heterostrophus at 4 h after inoculation, Nomarski optics. (b) Germinated conidium of C. heterostrophus at 6 h after inoculation. Note the underlying host cells are pigmented red due to accumulation of deoxyanthocyanidin phytoalexins. (c) Between 4 and 6 h many host cells have developed inclusions (arrow) that surround the tip of the conidium. Inclusions also line one edge of the conidium, and similar spheres are on the upper surface of the conidium. The inclusions shown here are not yet pigmented, Nomarski optics. (d) A high concentration of, red colored phytoalexin inclusions surround an ungerminated conidium at 9 h after inoculation. Figures (d), (e), and ( f) are of conidia in suspension. (e) and ( f) By 20±24 h numerous round, red phytoalexin inclusions have accumulated around both germinated and ungerminated conidia, respectively. (g) Germinated conidium on the surface of a mesocotyl. Note the intense pigmentation of the mesocotyl and collapse of the germ tube.
At 6 hpi both the host tissue and the conidia began to change color, and conidia appeared less healthy than those seen at earlier stages after inoculation. At 10 hpi,
a yellow color was evident in host tissue where germinated conidia had attempted to penetrate. Thus they looked like Fig. 3(a). Chemical identi®cation of the yellow material was not attempted yet the yellow color would seem to infer that the material might be ¯avonoid in nature. Consistent with this, an increase in luteolinidin content in the plant extract was also detected by HPLC (Fig. 2). At 16 hpi both the host tissue and the conidia appeared red and looked like Fig. 3(b). It was clear that the highest accumulation of phytoalexins occurred at sites where the fungus originally attempted to penetrate (Fig. 3(b)). Appearing at 9 hpi and very evident after 20 hpi, a high concentration of round, red colored phytoalexin inclusions was observed in samples and the majority of these inclusions had accumulated around conidia [Figs 3(d)±(g)]. At 24 hpi the intense red coloration in the host tissue and the pathogen was due to a high concentration of phytoalexins in inclusions and associated with conidia [Figs 3(d)±(g)]. The conidia appeared to have taken up the compounds resulting in conidial death as indicated by collapse and dehydration of germ tubes (Fig. 3(g)). At 38 and 40 hpi many phytoalexin inclusions surrounded dead conidia and both the inclusions and the conidia were of the same intense red color and looked like Figs 3(d)±(g). This observation coincided with high concentrations of luteolinidin and apigeninidin in plant extracts (Fig. 2). The accumulation of phytoalexin inclusions was typically higher at the ends of the conidia, the sites from which germination occurred [Figs 3(d) and (e)]. Observations of germinated conidia at 40 hpi showed that the highest concentrations of inclusions were around germ tubes, as in Fig. 3(e). It is known that substantial quantities of extracellular material are released as part of the fungal germination process [8, 12]. Based on these observations, we propose that phytoalexin inclusions are `trapped' by extracellular sheath material that conidia release during the germination process (Fig. 3(e)). It is possible that this is the site where the 3-deoxyanthocyanidin compounds initiate their toxic eects.
DISCUSSION Accumulation of 3-deoxyanthocyanidins showed the same behavior as that of previous work [2]. The contribution of this work to the understanding of this resistant response is that we detected, by HPLC analysis, that the 3-deoxyanthocyanidin compounds were released hours earlier than previously assumed [2]. Thus, we may infer that host tissue can rapidly detect the presence of the pathogen and as soon as the fungus starts to attempt penetration the host initiates its defences.
Interaction of Cochliobolus heterostrophus with phytoalexin inclusions in Sorghum bicolor The presence of free inclusions in water indicates that these phytoalexins are not as water soluble as previously thought [11]. It is also possible that the inclusions are derived from the ER-Golgi complex and remain membrane-enclosed. The composition of phytoalexins in the inclusions may de®ne inclusion color. The release of these compounds seems to depend on time after inoculation (Fig. 2). At 10 hpi several inclusions were composed primarily of luteolinidin and apigeninidin (Fig. 2) and this could account for host tissue color. HPLC analyses allowed us to detect the 3-deoxyanthocyanidins earlier than expected. The sensitivity of HPLC analyses may explain the dierence between our detection of phenols from 0 to 8 hpi and earlier reports. Thus, accumulation of phytoalexins in the sorghumC. heterostrophus interaction occurs much more rapidly than previously thought. It is also clear that phytoalexin inclusions become associated with the extracellular mucilage produced by the pathogen during germination.
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