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Effect of tissue wounding and timeof soil inoculation on pepper root rotdevelopmentS. E.L. Alao a , O. Alabi a , A. D. Akpa a , M. D. Alegbejo a & P. S.Marley aa Department of Crop Protection , Institute for AgriculturalResearch, Ahmadu Bello University , P.M.B. 1044, Zaria , NigeriaPublished online: 11 Jul 2011.

To cite this article: S. E.L. Alao , O. Alabi , A. D. Akpa , M. D. Alegbejo & P. S. Marley (2012)Effect of tissue wounding and time of soil inoculation on pepper root rot development, Archives OfPhytopathology And Plant Protection, 45:4, 468-474, DOI: 10.1080/03235408.2011.587985

To link to this article: http://dx.doi.org/10.1080/03235408.2011.587985

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Effect of tissue wounding and time of soil inoculation on pepper root

rot development

S.E.L. Alao*, O. Alabi, A.D. Akpa, M.D. Alegbejo and P.S. Marley

Department of Crop Protection, Institute for Agricultural Research, Ahmadu Bello University,P.M.B. 1044, Zaria, Nigeria

(Received 16 January 2011; final version received 5 March 2011)

Five-week-old pepper plants with wounds created on stems and roots weretransplanted to soils having inoculum of Phytophthora capsici incorporated fordifferent lengths of time. Disease severity (39.99%) on root trimmed seedlings wasnot significantly different (P � 0.05) from the severity (36.24%) obtained on stemlacerated seedlings. The wound treatments did not result in significantly differentrates of lesion extension per day; stem lacerated seedling had the fastest, 1.99 mm/day lesion extension rate, followed by 1.90 and 1.89 mm/day extension ratesobtained on root trimmed and unwounded treatments, respectively. However,time of soil inoculation had significant effect on severity; root trimmed and stemlacerated treatments had 46.3% and 39.8% severities, respectively. Tissuewounding 6 time of soil inoculation interaction did not have significant effecton disease severity; stem lacerated seedlings transplanted to 1-day and 3-dayinoculated soils gave highest severity (49.9%), followed by seedlings inoculated atthe time of transplantation. Root trimmed seedlings inoculated at the time oftransplantation had highest severity (61.1%), while the lowest severity wasobtained on seedlings transplanted to 5-day inoculated soil.

Keywords: stem laceration; root trimmed; disease severity; Phytophthora capsici;Capsicum annuum

Introduction

The peppers, Capsicum annuum (L.) and Capsicum frutescens (L.), are importantcrops grown as annual or biennial shrubs in all parts of Nigeria; large scaleproduction is, however, from the northern parts (Olarewaju and Mohammed 1990).Despite being Africa’s leading producer, 723,000 tonnes produced per annum (FAO2007), the 793 kg/ha yield is very low due to a plethora of pests and diseases (Boslandand Votava 1999). The incompetent method of transplanting pepper seedlings withvery high percentage of either immature or injured seedlings also contributes to thelow yield (Adedoyin 1990).

Kim et al. (1990) observed that disease severity was increased on fields infectedwith Phytophthora capsici, cultivated in Southern provinces of Korea, when injurieswere present on pepper roots, stems, fruits and foliage. Biles et al. (1992) reportedthat although P. capsici does not require wounding of host tissues for diseasedevelopment, severity of disease significantly increased whenever wounds

*Corresponding author. Email: alaoemma@gmail.com

Archives of Phytopathology and Plant Protection

Vol. 45, No. 4, February 2012, 468–474

ISSN 0323-5408 print/ISSN 1477-2906 online

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/03235408.2011.587985

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(mechanical injuries) occurred. Of recent, an identified disincentive to pepperproduction in northern Nigeria is the pepper root rot pathogen, Phytophthora capsici(Leon.), whose post-harvest survival in soil is accomplished by oospores with cellwall composed of multilayered b-glucan and cellulose (Alegbejo et al., 2006). Theoospores are hyaline, slightly wrinkled, spherical (between 25 and 35 mm in diameter)with 2.0–2.5 mm thick walls (Waterhouse 1963), and they remain infective in fallowsoils up to 10 years (Lamour and Hausbeck 2002), although germination can occuronly after a month of dormancy (Hausbeck and Lamour 2004). The produced germtubes can penetrate fresh plant tissues and the successful infection results in therelease of biflagellate zoospores that swim briefly before encysting. The duration offree water in soil, on foliage and on fruit is important in the development of pepperroot rot because the zoospores of P. capsici can only proliferate and cause infectionin the presence of free water (Wilcox and Mirecetich 1985).

Manohara et al. (2002) reported that P. capsici infected propagules survivedmore than 20 weeks in latosol soil at 100% field capacity, with some Phytophthoraspp. surviving saprophytically on pepper leaves for 11 weeks in soils held at 60–100% field capacity. This study was conducted at Samaru (118 110N 078 380E, at686 m above sea level) to determine the effect of transplanting injured seedlings tosoils contaminated with P. capsici on disease severity.

Materials and methods

Five-week-old seedlings of local variety SAMPEP 4 sown in sterilised soil(loam þ washed river sand in a 1:3 ratio) were transplanted to earthen pots(20 cm diameter) which had N:P:K (15:15:15) at 135 kg/ha thoroughly mixed in. Thethree wound treatments, namely root trimmed, stem laceration and not woundedwere made as described.

Seedling roots were 5 mm trimmed using flame sterilised scissors; while fivelacerations (each approximately 5 mm long) were made at collar region with flamedrazor blade before transplantation. The wounds were made with stems of seedlingsplaced in sterile water. Seedlings that had no wound made were inoculated with P.capsici. Check plants had appropriate wounds made and were inoculated with steriledistilled water (SDW).

An isolate of P. capsici obtained from the Department of Crop Protection,Ahmadu Bello University, Zaria, Nigeria was grown on oatmeal agar in Petri dishesincubated at 28 + 0.58C for 5 to 7 days. The culture of P. capsici was flooded with10 ml of SDW and chilled for 2 h at 48C to induce the release of zoospores. Myceliawere harvested, macerated using low speed Warren blender and decanted throughdouble layer cheesecloth. Released zoospores were counted using haemocytometerand final concentration was adjusted to 2.5 6 105 zoospores/ml. Twenty millilitresof the suspension of motile zoospores were used to drench soil contained in each pot.SDW (250 ml) was used to flood each pot prior to transplanting seedlings. Inoculumof P. capsici was applied to drench sterilised soil 0, 1, 2, 3, 4, and 5 days beforetransplanting a five-week-old seedling. Treatment where P. capsici inoculum wasapplied at transplanting time represented the 0-day treatment (T1). Check wasinoculated with only SDW before seedling was transplanted.

One hundred and eighty earth pots were arranged in a completely randomiseddesign (CRD) on the screen house bench. Each treatment was replicated five times.

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Parameters taken include incubation period, lesion length on stem and seedlingmortality at 21 days after transplanting (DAT). Rate of lesion extension per day wascalculated as:

Rate of lesion extension ¼ Length of lesion at tn 7 Length of lesion at t1,

where tn ¼ measurement on final day and t1 ¼ measurement on day 1.Disease severity was rated daily after inoculation based on a 1–9 rating scale

described by Adorada et al. (2000) in which 0 ¼ no symptoms; 1 ¼ small, irregularwater-soaked lesions on the upper leaves, leaf chlorosis; 3 ¼ leaf wilts; 5 ¼ brownishmargined lesions appearing on stem; 7 ¼ lesions on stem extending �10 mm abovesoil level, stem girdled; and 9 ¼ entire plant death. Disease severity was calculatedusing formula described by Adorada et al. (2000).

Disease severity ¼ Sum of rating scores

Number of plants assessed �Maximum severity score� 100:

Data were analysed using the Statistical Analysis Systems (SAS 1993) and significantmeans was separated using the Student–Neuman–Keuls (SNK) test. The experimentwas repeated twice.

Results

The effect of tissue wounding on incubation period was not significant (P � 0.05),2.89 days, being the shortest incubation period, was obtained on root trimmedtreatment followed by 3.32 days on stem laceration treatment. Treatment whereseedlings were not wounded had longest incubation period, 3.79 (Table 1). Thewound treatments did not result in significantly different rates of lesion extension per

Table 1. Effect of tissue wounding and time of soil inoculation with P. capsici on pepper rootrot development.

TreatmentIncubationperiod (days)

Stem lesion(mm/day)

Mortality at21 DAI (%) Disease severity (%)

Stem laceration 3.32a 1.99a 99.4a 36.24a

Root trimming 2.89a 1.89a 100a 39.95a

Not wounded 3.79a 1.90a 100a 39.95a

SE (+) NS NS NS NSSoil inoculation (days)0 4.00ab 3.10a 82.50a 52.8a

1 3.63ab 2.17b 77.50a 47.2ab

2 3.75ab 2.52ab 65.00a 44.4ab

3 4.25ab 1.82bc 55.00a 41.7ab

4 4.25ab 1.57bc 47.50a 33.3ab

5 5.00a 1.75bc 63.75a 38.9ab

Means 3.55 2.15 55.89 43.05SE (+) 3.83 2.25 NS 0.7Interaction (W 6 D) NS ** NS NS

a,b,c ¼ means followed by the same letters in the same column are not significantly different (P � 0.05);NS ¼ not significant; DAI ¼ days after inoculation; interaction (W 6 D) ¼ wound 6 soil inoculationinteraction.

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day; stem lacerated seedling had the fastest, 1.99 mm/day lesion extension rate,followed by 1.90 and 1.89 mm/day extension rates obtained on root trimmed andunwounded treatments, respectively. Irrespective of the wound treatments, plantmortality 21 days after inoculation (DAI) was high (100%). Although, woundingtreatment did not result in significantly different severity (P � 0.05), the highestseverity 39.99% was obtained on root trimmed and unwounded seedlings, the lowestwas on stem lacerated treatment.

Time of soil inoculation had a highly significant effect (P ¼ 0.05) on incubationperiod, with incubation period 5.00 days being the highest on seedlings transplantedinto 5-days inoculated soil followed by 4.25 days recorded for seedlings transplantedinto 3- and 4-days inoculated soils. The shortest period 3.63 days was on seedlingstransplanted in 1-day inoculated soil (Table 1). Irrespective of the soil inoculation time,plant mortality at 21 DAI was not different. The highest mortality 82.50% wasobtained where inoculum was placed at the time of transplanting the seedlings.Differences of 6.06% and 16.13% in plant mortality was obtained as time of soilinoculation increased from 0 day to 4 days but plant mortality increased 25.49%between 4- and 5-day soil inoculation. The effect of soil inoculum placement wassignificant on rate of lesion extension per day, with fastest extension rate produced onseedlings treated at 0-day inoculation followed by 2-day inoculation treatment. Rate oflesion extension for the 1-, 2-, 3-, 4- and 5-day treatment were at par statistically.

The main effect of soil inoculation on disease severity was significant (P � 0.05),seedlings transplanted and simultaneously inoculated had highest severity, 52.8%.Severity 33.3% on seedlings in the 4-day soil inoculation was the lowest. Reductionin disease severity as number of days soil inoculum was placed increased up to 4 daysbefore an increase was obtained.

Interaction effects of tissue wounding 6 soil inoculation was not significant onincubation period, mortality 21 DAI and disease severity (Table 1). The interactionof stem laceration 6 soil inoculation treatment did not affect rate of lesionextension/day; rate of lesion extension decreased with subsequent increase in soilinoculation days (Table 2). This same trend was observed for the seedlings that werenot wounded. However, rate of lesion extension was significantly different(P � 0.05) for the root trimmed 6 soil inoculation interaction; the highest ratesof extension were obtained in seedlings placed in soils inoculated 3, 5 and 0 day,respectively, followed by 4 days placement while least rate of extension was on the2-day soil inoculated treatment.

Discussion

Bostock and Stermer (1989) defined the removal or alteration of physical barriersbetween a pathogen and the internal structures of a living plant as wound. That pepperroot rot development was not significantly increased by stem lacerations or roottrimming was clearly demonstrated in this study as was reported by Biles et al. (1992).

Incubation period needed before disease symptoms become visible, plantmortality occasioned by infection with P. capsici as well as disease severity oramount of disease present on the tissues were appropriate parameters to indicate thatthe pepper root rot pathogen does require extended time length for a contaminatedsoil to become an avenue for infection. Reductions in the amount of disease andmortality of plants in infected soils were observed as age of the pathogen increased inthe soil, a situation that was similarly reported by Zitter (1989). Where continuous

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Table

2.

Woundbysoilinoculationtreatm

entinteractiononrate

oflesionextension.

Rate

oflesionextension(m

m/day)in

soilsinoculatedatday

Woundtreatm

ent

01

23

45

Stem

laceration

3.1

a+0.03

2.17a+

0.03

2.00ab+

0.05

1.80ab+

0.03

1.00b+

0.00

1.00b+

0.00

Roottrim

ming

3.1

a+

0.03

2.17a+

0.03

3.59a+

0.03

2.66a+

0.03

2.71a+

0.03

3.25a+

0.00

Notwounded

3.1

a+

0.03

2.17a+

0.03

1.57b+

0.05

1.00b+

0.03

1.00b+

0.00

1.00b+

0.00

SE

(+)

NS

NS

0.75

0.97

1.02

0.95

a,b¼

meansfollowed

bythesamelettersin

thesamecolumnare

notsignificantlydifferent(P�

0.05);NS¼

notsignificant.

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high humidity conditions exists, multiple cycles of sporangia production will lead tomany infection cycles within a crop season. Garrett (1975) noted that just a sterilesoil environment is artificial and does not exist in nature, and so the microbialpopulation dynamics within natural system is never static. Mitchell (1978) reportedthat Phytophthora spp., unlike other soil-borne pathogens, has limited saprophyticcapabilities in the absence of its host.

P. capsici was clearly shown to be highly pathogenic to the pepper roots assignificantly high disease severity was recorded on seedlings transplanted to soilswith 5-day inoculum. Compared to the stem lacerated and unwounded treatments,plants with injured roots specially attracted the pathogen with subsequent diseasedevelopment. These observations highlight the issues raised by Adedoyin (1990) andKim et al. (1990) who observed very high incidences of pepper root rot on fieldswhere farmers transplanted injured seedling or weakened seedlings. On the contrary,un-injured plants showed little signs of the disease when transferred to soils infectedabove 3 days with Phytophthora. Since it is known that conditions that aregenerally favourable for P. capsici development on the fields include wet soil withtemperatures above 188C or prolonged wet periods with air temperatures rangingfrom 248C and 308C (Duniway 1976; Erwin and Ribeiro 1996), farmers would betterdelay the transplantation of seedlings until at least a week after adequately preparingsoil. This period would ensure that the pathogen is destroyed by the exposure tounfavourable conditions occasioned by land tilling, weed destruction and consequentnon-availability of fresh host tissues to colonise. Bos et al. (1989) had reported thatstrictly adhering to crop rotation schedules particularly in the Fadama and dampareas would ensure reductions in populations of soil-borne pathogens.

Conclusions

P. capsici, being highly pathogenic in the presence of pepper roots, is capable ofproducing multiple cycles of sporangia within a single season that will lead to manyinfection cycles. Disease severity recorded on seedlings transplanted to soils havinginoculum 5 days earlier was significantly high and it was noted that where conditionsfor high humidity exists, development of pepper root rot would increase significantlyon seedling stem and roots having injuries.

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Alegbejo MD, Lawal A, Chindo P, Banwo OO. 2006. Outbreak of basal stem rot and wiltdisease of pepper in northern Nigeria. J Plant Prot Res. 46(1):7–13.

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