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Keywords:

  • Capsicum annuum;
  • infection studies;
  • peroxidase;
  • zoospores

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Greenhouse experiments were conducted to determine disease progression of phytophthora root rot on nonwounded and wounded pepper plants (Capsicum annuum) and to determine whether susceptibility to Phytophthora capsici decreases with wound aging. Two isolates of P. capsici were used in this study, one less aggressive than the other. Trimming the roots immediately prior to inoculation with either isolate increased susceptibility significantly (P ≤ 0·05) compared with plant roots that were not trimmed. Both isolates caused a higher level of disease severity on disturbed/trimmed than on disturbed/nontrimmed roots. Disease also occurred earlier with the more aggressive isolate on both wounded and nonwounded roots. Disease severity was three to four times more severe on plants treated with the aggressive isolate (NM6011) than on those inoculated with the less aggressive isolate (NM6040), regardless of root treatment. In separate experiments, pepper roots were wounded and allowed to age for up to 5 days before inoculation. Resistance to P. capsici increased as the wounds aged, resulting in significantly (P ≤ 0·001) lower disease severity on plants with 3- and 5-day-old wounds than on those inoculated at the time of wounding and the nondisturbed/nontrimmed controls. Wounding of the roots followed by immediate inoculation with zoospores resulted in significantly higher levels of attachment than when roots were inoculated with zoospores 48 h after wounding. The 48-h postwounding inoculation treatment showed the same amount of zoospore attachment as nonwounded roots. Increase in plant resistance correlated (P ≤ 0·01) with an increase in total peroxidase activity. Isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) indicated increased band intensity of three acidic and one basic isozyme as wounds aged. These data suggest that wound repair plays a role in decreasing infection and resultant disease symptoms of pepper to P. capsici.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Phytophthora root rot and blight of pepper, caused by Phytophthora capsici, is one of the most economically destructive soil-borne diseases of pepper (Capsicum annuum) throughout the world. The most common symptoms associated with the disease are wilting and a root and crown rot characterized by a dark brown stem lesion extending upward from the soil line (Black et al., 1991). The advancing lesion eventually girdles the main stem and kills the plant (Bowers & Mitchell, 1991).

In New Mexico, phytophthora root rot and blight occurs on the roots, stems, leaves and fruit (Shannon, 1989; Black et al., 1991; Wall & Biles, 1993) of long green, jalapeño and cayenne peppers, and severely reduces yield and fruit quality. P. capsici is also pathogenic on tomato, eggplant, cucumber, watermelon, pumpkin, squash, cocoa and macadamia (Leonian, 1922; Kreutzer et al., 1940; Kunimoto et al., 1966; Polach & Webster, 1972; Ristaino, 1990).

In pepper-growing fields, standard field operations and cultural practices may contribute to increased susceptibility of the plant to the pathogen resulting in a higher level of disease. A survey conducted by Kim et al. (1990) in Suwon, South Korea, indicated that plants receiving numerous injuries due to cultivation developed the highest incidence of P. capsici blight. Wounding of roots often occurs during transplantation and cultivation. Phytophthora root rot of New Mexican-type peppers has been observed to increase immediately after cultivation (P. W. Bosland, personal communication). Even though P. capsici does not require a wound for ingress, predisposition of the host by wounding has been shown to enhance chemotactic attraction and encystment of zoospores to the wound location (Carlile, 1983; Waugh et al., 1993). After encystment Phytophthora zoospores germinate and directly penetrate host tissue with the aid of enzymatic degradation of the plant cell wall (Coffey & Wilson, 1983).

Wounds can be generated by numerous agents including severe weather (e.g. wind, hail, freezing), insects, large herbivores, human activities (e.g. cultivation) or simply during the normal developmental processes of the plant (e.g. abscission, growth cracks, lateral root emergence) (Bostock & Stermer, 1989). In response to wounding, peroxidase has been reported to play a role in lignification, suberization, polymerization of polyphenols and in stimulating other defence mechanisms (Hammerschmidt et al., 1982; Kolattukudy et al., 1992). In addition, peroxidase also has been reported to have antifungal activity (Peng & Kuc, 1992).

The purpose of this study was to determine if damage to pepper roots significantly increased the severity of phytophthora root rot and if age of wound was a determinant in plant susceptibility. Assays were conducted to determine whether peroxidase activity and isozymes increased with increasing wound age and could be correlated with disease resistance. In addition, microscopic studies of wounded roots inoculated with P. capsici zoospores were conducted to determine whether age of wound affected zoospore attachment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Plant material and inoculum preparation

Phytophthora capsici was isolated from two different locations in New Mexico, USA: isolate NM6011 in Las Cruces and isolate NM6040 in Anthony. The isolates were recovered from symptomatic plants in chile pepper crops and maintained on corn meal agar plates. Preliminary experiments showed that isolate NM6011 was more aggressive than NM6040 (Biles et al., 1991). The procedure described by Ristaino (1990) was used to promote zoospore production, except that the V-8 agar discs with P. capsici hyphae were incubated in sterile soil extract at 27°C. Sporangia were treated with a 1-h cold shock (6°C) followed by a 1-h equilibration at room temperature. Zoospore suspensions were calibrated with a haemocytometer.

Chile peppers (cv. New Mexico 6–4) were sown in 4-cm-diameter cells (Com-Packs, TO Plastic Inc., Minneapolis, MN, USA) with commercial potting soil (Metro-Mix 360 Growing Medium, Grace Sierra Horticultural Products Co., Yosemite Drive, Milpitas, CA, USA) at a rate of one plant per cell and maintained in a greenhouse. Plants were watered daily as required to keep the soil moist. Plants were grown for 4–5 weeks (four-leaf stage). Roots were inoculated by immersion in 200 mL of an inoculum suspension at a concentration of 1000 zoospores per mL of sterile distilled water for 24 h. Control treatment was provided by immersing roots in sterile distilled water alone.

Effect of root wounding

To determine the effect of root wounding on susceptibility to P. capsici, plants were subjected to two wounding treatments: disturbed/nontrimmed, which involved carefully lifting the plant from the soil with minimal disruption of the roots and returning it to the soil; and disturbed/trimmed, in which the bottom 2 cm of the root system was trimmed off with a pair of flame-sterilized scissors after washing off the soil and the plant was placed back into the soil. Plants were either inoculated or not inoculated on the same day with one of the two isolates of P. capsici as described above. Nontrimmed and nondisturbed plants (plants not removed from the container) were inoculated as controls. Four replicates were used, with six plants per replicate.

Disease severity was evaluated each day after inoculation (DAI) by scoring individual plants using a 1–9 scale. The scale was based on the area or length of stem base affected by disease relative to the total plant height, where 0 = no infection, 1 = wilting or yellowing, 2 = 1–5% lesion, 3 = 6–25%, 4 = 26–50%, 5 = 51–60%, 6 = 61–70%, 7 = 71–80%, 8 = 81–90%, and 9 = 91% and above. Disease severity expressed as a proportion (in percentage) of the total plant height was computed using the following formula:

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Effect of time of wounding prior to inoculation

To determine the effect of wound age on susceptibility of pepper roots, plants were wounded by disturbing/trimming the roots 0, 1, 2, 3 or 5 days before inoculation. The more aggressive isolate (NM6011) was used in this experiment at a concentration of 1000 zoospores per mL. Four replicates were used with six plants per replicate. The experiment was performed twice using a completely randomized design. The number of days taken for symptoms to develop was recorded for each treatment, and disease severity was evaluated after inoculation as described above.

Zoospore attachment to wounded roots

Pepper seedlings were grown as described above, except that vermiculite was used instead of potting soil. Wounding of the roots was accomplished by excising roots of 12-week-old pepper plants (12 weeks from sowing) 5 cm below the crown at 0, 48, 96 and 120 h prior to inoculation with either NM6011 or NM6040. Following wounding, all plants except for the 0-h time treatment were repotted in vermiculite and maintained under normal greenhouse conditions until inoculation. Nonwounded plants were used as controls at each time period. Inoculation was carried out in 30-mL sterile glass tubes with a 1 × 105 zoospore per mL suspension. The roots were incubated at room temperature (25°C) for 1 h, and then rinsed gently in sterile distilled water. In order to detect zoospores encysted on the root surface, a solution of 0·05% Rose Bengal in 70% ethanol was applied to the roots for 16 h at 4°C. Roots were destained with 70% ethanol for 30 min. Observations were made at ×400 magnification and all attached zoospores in one field of view at the wound site were counted. Each experiment consisted of four replicate plants per treatment, with three subsampled roots per replicate.

Peroxidase assays of root tissue

Chile peppers were grown in potting soil as previously described. The roots were trimmed 5 cm below the top lateral root near the crown approximately 0, 6, 24, 48 and 96 h before assaying the tissue for peroxidase activity. The distal 2 cm of trimmed root tissue was taken from each plant, weighed, and ground with a mortar and pestle in Tris-HCl 50 mm, pH 7·5 buffer [1 : 2 (w:v.) tissue:buffer] using sterile quartz sand to aid cell disruption. The slurry was transferred to a microcentrifuge tube and centrifuged for 5 min at 12 000 g. The supernatant was placed in a fresh tube and used immediately in assays or stored at − 20°C.

Spectrophotometric readings (470 nm) were taken 1 min after 25 µL of the root extract was added to 3 mL of 10 mm guaiacol and 10 mm H2O2 substrate in 50 mm acetate buffer, pH 5. Each plant represented a replicate and each treatment (wounding time) contained three or four plants.

Isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) was used to separate root peroxidase isozymes. Root extracts from individual plants were placed on Whatman #1 wicks (5 × 5 mm) (Whatman International Ltd., Maidstone, UK). The wicks were placed in the middle of a 5% T, 3·3%C IEF-PAGE with a 3·2–9·7 ampholyte gradient. The gel was electrophoresed for 20 min at a constant 100 V, the wicks were then removed, and the gel was run for an additional 1 h at 350 V. Except for a modified run time, the procedures of the manufacturer were followed (Model 111 Mini-IEF Cell, Bio-Rad Laboratories, Richmond, CA, USA). Immediately after removing the gel from the electrodes, the gel was stained for peroxidase activity with 10 mm guaiacol and 10 mm H2O2 in 50 mm acetate buffer, pH 5. Isoelectric points were determined by using Bio-Rad broad range IEF standards (pI 4·6–9·6).

Statistical analyses

Statistical analyses were performed with the Statistical Analysis System (SAS Institute, Cary, NC, USA). Data were subjected to a two-way analysis of variance (anova). Treatment means were compared with Dunnett's minimum significant difference test or Fisher's protected least significant difference test. Regression analyses were performed on the following data sets: disease severity against days after inoculation for each treatment within isolate; disease severity against age of wounds; and peroxidase activity against time after wounding. Several correlations were performed on disease severity against peroxidase activity (time + 0 days; time + 1 day; time + 2 days; and time − 1 day) to detect time delay effects. All experiments were conducted three times unless otherwise indicated.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Effects of wounding experiments

Disease symptoms were apparent on the stem of plants 5 days after inoculation (Table 1) and were characterized by dark brown lesions extending upward from the soil line. Plants that were wounded but not inoculated did not develop symptoms.

Table 1.  Disease severity caused by two isolates of Phytophthora capsici on chile pepper roots
 Days after inoculationa
Isolate5b678910
  1. a Percentage disease severity was evaluated using a 1–9 scale where percentage of plant tissue affected by disease was expressed as a proportion of total plant height. Data are the means for each isolate (factor one) regardless of wounding treatments (factor two) in a two-factor experiment based on four replicates. No significant interaction between isolate and wounding treatments was detected. b Symptoms were not observed until 5 days after inoculation. c Means in each column are significantly different (P = 0·05) based on Fisher's least significant difference test . Data from wound treatments (disturbed/trimmed, disturbed/nontrimmed, nondisturbed) were combined (shown in Fig. 1).

NM 60114·7815·4640·7458·0266·8282·87
NM 60400·002·4712·9529·9446·4559·38
LSDc2·715·009·7013·3614·5915·23

Plants that were wounded and inoculated with either isolate showed increased disease severity as time progressed (Fig. 1). Regression analyses were performed on data for disease severity 5–10 DAI, and regression equations were significant and best fit a linear model (P ≤ 0·0001). The linear equations described the increase in disease severity with time (DAI) and indicated that for both isolates disturbed/trimmed plants had the highest rate of disease progression, followed by the disturbed/nontrimmed plants and then by the control (plants not removed from container). Disturbed/trimmed plants consistently had higher (P = 0·05) disease severities than the control from the day of symptom initiation, 5 DAI, to 10 DAI. Mean disease severity of the two isolates at 5 DAI was 4·86 and 0·70% in the disturbed/trimmed and control plants, respectively. Final mean disease severity was 88·62 and 55·56% at 10 DAI in the disturbed/trimmed and control plants, respectively. Disturbed/nontrimmed plants were not significantly different (P ≤ 0·05) from the control. Greater disease severities were observed in plants inoculated with isolate NM6011 than with isolate NM6040 at all sampling dates (Table 1). The interaction between wounding and isolate was not significant.

image

Figure 1. Disease severity of Phytophthora root rot 1–10 days after inoculation (DAI) of roots that were disturbed/trimmed, disturbed/nontrimmed or nondisturbed/nontrimmed (control). Two different isolates of Phytophthora capsici were used: (a) NM6011 and (b) NM6040. The data best fit a linear regression model with the following equations: Y = − 64·8 + 12·8X (R2 = 0·71), Y = − 81·0 + 16·6X (R2 = 0·88) and Y = − 81·0 + 18·8X (R2 = 0·92) for the control, disturbed/nontrimmed plants and disturbed/trimmed plants, respectively, inoculated with isolate NM6011. For isolate NM6040, the regression equations were Y = − 59·0 + 103X (R2 = 0·71), Y = − 57·3 + 10·2X (R2 = 0·55) and Y = − 94·7 + 17·7X (R2 = 0·86) for the control, disturbed/nontrimmed plants and disturbed/trimmed plants, respectively. Y represents percentage disease severity and X represents days after inoculation for both isolates.

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Effects of root wounding at different time intervals before inoculation

Not all plants showed symptoms at 5 DAI, but by 11 DAI the majority of the plants showed symptoms. Therefore, disease severities were obtained for each treatment and then compared as an effect of age of wound at inoculation on severity of the disease. Percentage disease severity decreased linearly (P ≤ 0·0001) as age of wounds increased. The equation Y = 72·41–15·24X (R2 = 0·59) represents age of wound at inoculation (X) and the percentage disease severity (Y). Mean disease severity was 84·7, 54·1, 33·3, 14·8 and 7·4% on peppers wounded 0, 1, 2, 3 and 5 days before inoculation, respectively. Mean disease severities of wounds aged for 1–5 days before inoculation were significantly different (P ≤ 0·05) from the control (0 days before inoculation) according to Dunnett's one-tailed t-test.

Disease severity decreased significantly (P ≤ 0·001) as the interval between inoculation and wounding increased from 0 to 5 days. The number of days to symptom appearance increased as age of wound increased. Pepper plants with 0–1-day-old root wounds showed symptoms 4–6 DAI, while plants with 2–5-day-old root wounds showed symptoms 4–12 DAI. Logistic regression analysis showed that the probability of number of days to symptom appearance was positively correlated (χ2 = 17·59, P > 0·00003) with the age of wound. A cumulative logistic probability plot of symptoms being present on particular days depending upon age of wound at inoculation is shown in Fig. 2. The probability of symptoms appearing at 0–5, 6–8 or 9–11 days is partitioned such that the total probability of symptoms appearing is always 1·0. Two logistic regression equations describe the boundary probabilities between each class of days to symptom appearance as follows. The line describing the boundary probability (P) that root rot symptoms will appear at either 0–5 days or 6–8 days is described by:

image

Figure 2. Relationship between age of wounds (disturbed/trimmed roots) at inoculation and number of days to symptom appearance of Phytophthora root rot. The regression lines were best described by logistic probability curves expressed as: ln[P/(1 − P)] = − 1·7090 + 1·9176 × (number of days to symptom appearance × age of wound) and ln[P/(1 − P)] = − 1·7090 + 3·7550 × (number of days to symptom appearance × age of wound). R2 for the whole model was 0·42. X represents age of wound at inoculation and Y represents the probability (P) that root rot symptoms will appear on a particular root wound after a certain number of days defined by the relationship: P = 1/{1 + eln[p/(1–p)]}. Points on the plot in the midpoint of the probability interval are actual responses.

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inline image

The line describing the boundary probability (P) that root rot symptoms will appear at either 6–8 days or 9–11 days is described by:

inline image

Zoospore attachment on wounded roots

Attachment of zoospores was significantly higher in roots inoculated immediately after wounding than in those inoculated 48, 96 and 120 h later (Fig. 3) and in nonwounded controls. At 0-h wound sites, attachment of NM6011 was four-fold greater than that of the less virulent isolate NM6040. Attachment of zoospores to 48-, 96- and 120-h-old wounds was not significantly different from nonwounded controls (data not shown).

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Figure 3. Attachment of Phytophthora capsici zoospores to disturbed/trimmed roots of pepper. NM6011 (highly aggressive isolate) or NM6040 (less aggressive isolate) was applied at 0, 48, 96, or 120 h after the roots were disturbed and wounded. Vertical bars represent standard error of the mean.

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Peroxidase activity of wounded roots

Peroxidase activity increased as age of wound increased (Fig. 4). An increase in the total peroxidase activity was evident 2 days after wounding and continued to increase up to 4 days. In a separate experiment, peroxidase activity started to decline at 5 days after wounding (data not shown).

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Figure 4. Peroxidase activity of chile pepper roots 0–4 days after wounding. Data were combined from two separate experiments. Vertical bars represent standard error of the mean.

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Acidic and basic peroxidase patterns were enhanced as the age of wound increased (Fig. 5). At 0 days, one acidic peroxidase band was detected at isoelectric point (pI) 4·6. As the age of wound increased additional isozymes became apparent and increased in intensity at the same putative locus. At 6 h, two acidic isozymes were detected at pI 5·0 and 6·0, and at 2 days, one basic isozyme was detected at pI 9·0.

image

Figure 5. Peroxidase isozymes of disturbed/trimmed pepper roots after 0–4 days. Isozymes were separated according to isoelectric point (pI) in an IEF-PAGE. Each lane was loaded with 3 µg mL−1 of total protein and stained with 10 mm guaiacol and 10 mm H2O2 immediately after electrophoresis. pIs were calculated with Bio-Rad IEF marker.

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Correlation analysis

A significant (P ≤ 0·01) inverse relationship between total peroxidase activity and disease severity was detected by correlation analyses of pooled data sets from two peroxidase activity experiments and two age-of-wound experiments. The correlation coefficient value of − 0·57 indicated that an increase in total peroxidase activity corresponded to a decrease in disease severity 1 day later.

Discussion

Many diseases are caused by pathogens that gain entrance through wounds (Bostock & Stermer, 1989). Even though P. capsici does not require wounds to infect, mechanical injuries rendered pepper roots more susceptible to the pathogen. By removing the physical barrier between the pathogen and the internal tissues by wounding, plants became more susceptible regardless of the aggressiveness of the isolate (Fig. 1 and Table 1). Similar results were observed with pepper fruit inoculated with P. capsici. Wounding was not necessary for disease development, but it significantly increased disease severity (Biles et al., 1993). Johnson & Miliczky (1993) also showed that more Colletotrichum coccodes lesions developed on potato leaves that were wounded than on leaves that were not wounded just before inoculation. These results indicate that injuries (wounds) on root, fruit and foliage will increase the severity of disease caused by various pathogens (Kim et al., 1990). In contrast, many of the chemical and structural barriers associated with expression of resistance to pathogens are also induced by mechanical injury (Mullick, 1977; Aist, 1983). One of these wound-induced responses that reduce pathogen ingress is wound repair.

Wounding of the roots followed by immediate inoculation (0 h) with zoospores of either isolate increased attachment of zoospores to the roots when compared with nontrimmed/disturbed roots and roots wounded 48, 96 and 120 h prior to inoculation. The decrease in zoospore attachment as the period between wounding and inoculation increased suggests that wound healing processes were occurring. NM6011, the highly aggressive isolate, showed significantly higher attachment immediately after wounding than isolate NM6040, which may account for its more aggressive nature on pepper. In all cases there was a pronounced time effect with significantly higher attachment to 0-h wounded plants than to plants wounded 48, 96 and 120 h prior to inoculation. Chemotaxis of Phytophthora sp. zoospores has been well documented (Carlile, 1983). Various root exudates attract Phytophthora zoospores, including several individual amino acids, sugars, alcohols, fatty acids and aldehydes (Carlile, 1983). Rapid accumulation and attachment of zoospores is usually observed just above the root tip in the zone of elongation on hosts and nonhosts in a similar fashion. However, the attraction of Phytophthora zoospores has also been shown to be more widespread over the root or limited to wounds (Carlile, 1983). Preliminary laboratory studies have shown similar results in regard to attachment to the zone of elongation. In addition, attachment of P. capsici zoospores to pepper roots was five-fold higher than on either tomato or cucumber (Waugh et al., 1993).

Freshly wounded pepper roots were more susceptible to P. capsici, and susceptibility decreased until wounds became resistant to the pathogen. These data suggest that as age of wound increases, resistance of pepper roots to the pathogen increases. The increase in resistance could be a result of a root wound repair process responsible for the build-up of physical and/or biochemical barriers associated with expression of resistance. In the present work, an increase in resistance correlated with an increase in peroxidase activity, in which acidic and basic peroxidase patterns were enhanced as the age of wound increased. The fact that more peroxidase activity was detected in older wounds that were observed to be more resistant than fresh or younger wounds further supports the role of peroxidase in disease resistance in this host–pathogen system (Seevers & Daly, 1970; Hammerschmidt & Kuc, 1982; Kolattukudy et al., 1992; Peng & Kuc, 1992; Biles et al., 1993). The present result suggests that isoperoxidases may be involved in the enhanced resistance to P. capsici in pepper roots. Peroxidases catalyze the oxidation of phenylpropane alcohols, generating free radical intermediates that combine nonenzymatically in a random fashion to form lignin. Lignin blocks the growth of pathogens in response to infection or wounding (Doster & Bostock, 1988; Taiz & Zeiger, 1991). Furthermore, it has also been shown that P. cinnamomi-resistant Eucalyptus spp. have a higher level of phenols and enzymes associated with lignification than susceptible plants (Cahill & McComb, 1992). Lignin has been implicated in the resistance of wheat to fungal pathogens (Vance et al., 1980; Reisner et al., 1986; Southernton & Deverall, 1990; Tiburzy & Reisener, 1990; Diaz & Merino, 1998), and more specifically to Phytophthora species (Doster & Bostock, 1988). The result of the present study also suggests that even though there was more peroxidase activity in aged wounds, disease development still progressed, although at a slower rate as the wounds aged. The role of peroxidase in the development of resistance in pepper plants to P. capsici is still unclear. However, there is a good correlation between increase in peroxidase activity and development of wound resistance (Hammerschmidt & Kuc, 1982; Kolattukudy et al., 1992; Biles et al., 1993), and between increase in peroxidase activity and lignin accumulation (Diaz & Merino, 1998). Vance et al. (1980) suggested that it is lignin that increases resistance of host cell walls to physical and enzymatic pathogen activity, thus inhibiting disease development.

In conclusion, wounded pepper roots were found to be more susceptible than nonwounded roots to P. capsici, regardless of the aggressiveness of the isolate. Fresh wounds were more susceptible, with susceptibility decreasing as wounds aged. In pepper fields it is a common practice to irrigate the field immediately after cultivation, transplanting or weeding. Phytophthora root rot is most severe when fields are over-irrigated (Biles et al., 1992). These results suggest that irrigation after cultivation should be delayed for at least 4–5 days. This would allow wounds or injuries incurred by the plant roots to heal and thus minimize disease damage. Initial field experiments comparing herbicide and cultivation treatments of pepper showed no significant effect on fruit yield when cultivation was minimized (Sollars et al., 1994). However, these experiments were conducted on naturally infested fields in which inoculum density and inoculum distribution were not known and irrigation regime was not considered. Further experiments need to be conducted on field plots with known inoculum levels. A comparison of irrigation applied immediately after cultivation with delayed irrigation would help determine the efficacy of this cultural control method.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

We thank Shane Drake and John Goldfarb for technical assistance.

This research was supported by the New Mexico Agricultural Experiment Station.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References
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Footnotes