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In vitro standardisation of resistancescreening methods in cassava againsttuber rot diseaseMuthukrishnan Senthil a , Vishnu Sukumari Nath a , MuthulekshmiLajapathyJeeva a , Vinayaka Mahabaleshwar Hegde a & Raj ShekarMisra ba Division of Crop Protection, Central Tuber Crops ResearchInstitute , Thiruvananthapuram , Kerala , Indiab Regional Centre for Central Tuber Crops Research Institute ,Bhubaneswar , Orissa , IndiaPublished online: 25 Feb 2013.

To cite this article: Muthukrishnan Senthil , Vishnu Sukumari Nath , Muthulekshmi LajapathyJeeva ,Vinayaka Mahabaleshwar Hegde & Raj Shekar Misra (2013) In vitro standardisation of resistancescreening methods in cassava against tuber rot disease, Archives Of Phytopathology And PlantProtection, 46:11, 1255-1261, DOI: 10.1080/03235408.2013.763618

To link to this article: http://dx.doi.org/10.1080/03235408.2013.763618

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In vitro standardisation of resistance screening methods in cassavaagainst tuber rot disease

Muthukrishnan Senthila, Vishnu Sukumari Natha, Muthulekshmi LajapathyJeevaa,Vinayaka Mahabaleshwar Hegdea and Raj Shekar Misrab*

aDivision of Crop Protection, Central Tuber Crops Research Institute, Thiruvananthapuram,Kerala, India; bRegional Centre for Central Tuber Crops Research Institute, Bhubaneswar,Orissa, India

(Received 7 December 2012; final version received 28 December 2012)

Cassava tuber rot caused by Phytophthora palmivora in growing regions of TamilNadu and Kerala, is causing yield loss up to 80%. In the present study, resistancereactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots byartificial inoculation method in search of a suitable in vitro resistant screeningmethod. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysisexhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analy-sis was superior in discriminating even small variations in resistance reactions thantuber analysis. The cultivar Sree Padmanabha showed higher resistance than othercultivars and the level of resistance in a cultivar is heritable which could be helpfulin breeding programme. Based on the results it can be concluded that leaves ofcassava could be used for screening resistance in the host and also in analysing thevirulence of the isolate. This is the first report on screening the resistance in cassavacultivars against root rot caused by P. palmivora.

Keywords: Phytophthora palmivora; tuber rot resistance; screening method;cultivars

Introduction

Cassava is widely cultivated in tropical and sub-tropical regions for its tuberous starchyroots by farmers for food purposes (Shore 2002) and also for its various industrial val-ues. In India, cassava is a cash crop in Tamil Nadu, Andhra Pradesh and is a staplecrop in regions of Kerala. Cassava production is hampered by many diseases caused bywide variety of pathogens. However, cassava root rot is one of the major diseases thatspread in most of the cassava growing countries. The organisms commonly reported tobe causing this destructive disease are Botryodiplodia theobromae (Onyeka et al.2005a), Phytophthora drechsleri, Rosellinia necatrix (Booth 1978; Theberge 1985),B. theobromae and Nattrassia mangiferae (Msikita et al. 1997). In addition to this,Phytophthora palmivora was reported from Southern India (Johnson and Palaniswami1999; Senthil et al. 2012b). After more than a decade, the same causal agent wasreported from China (Guo et al. 2012). It accounts for yield loss up to 80% and 30% inIndia and China, respectively. The disease is characterised by brown watery lesions in

*Corresponding author. Email: ctcritvm@yahoo.com

Archives of Phytopathology and Plant Protection, 2013Vol. 46, No. 11, 1255–1261, http://dx.doi.org/10.1080/03235408.2013.763618

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the infected tubers which emit a characteristic foul smell and rot within 5–7 daysdepending on the soil conditions. As a result, the tuber becomes unsuitable for furtheruse resulting in heavy economic loss. Since there are no visible symptoms at shootlevel in the infected plant, the disease is unnoticed until harvest. Rain and irrigated soilpromotes the spread of the disease. It is a serious pathogen in cassava growing regionsand the nature of the disease is poorly understood by the farmers. During our samplecollection, farmers reported severity of the disease in rainy season and the inefficacy ofthe disease control using biocontrol agent and fungicides. The reliable way to tacklethis problem is via breeding programmes for screening resistant cultivars (Hahn et al.1989) which is durable when external disease control measures fail.

Screening resistant cultivars depends on reliable results and the method adapted toassay (Vleehouwers et al. 1999) will be useful in identifying resistant/susceptible culti-vars. Carrying out tests in field is expensive, time consuming, may lead to wrong inter-pretation in case of non-uniformity of the pathogen in soil and also may cause outbreakof the disease or worsen the disease status. Various screening methods have beenreported earlier which use leaf, tuber or stem for analysing disease severity (Tahi et al.2000; Browne and Cooke 2004; Onyeka et al. 2005a; Vawdrey et al. 2005; Browneet al. 2006; Kunkeaw et al. 2010) and thereby sorting the resistant cultivars/germplasm.Onyeka et al. (2005a) evaluated the level of resistance using cassava tuber slice againstroot rot disease caused by B. theobromae and categorised cassava genotypes and germ-plasm based on disease response in vitro. Development of reliable resistant screeningmethod will be useful categorising resistant/susceptible genotypes and in large-scalescreening. So far, no reports are available on level of resistance in cassava cultivarsagainst tuber rot disease caused by P. palmivora.

Therefore, the main objective of this study was to identify a reliable screeningmethod for resistance against tuber rot disease based on in vitro inoculations in leaf,tuber and stem of cassava cultivars. This work may provide the basis for future studiesand is the first report on screening the cassava cultivars for resistant against tuber rotdisease caused by P. palmivora in India.

Materials and methods

Isolation of P. palmivora

Isolation of P. palmivora from infected cassava was performed according to our previ-ous report (Senthil et al. 2012b) and the isolate was maintained in carrot agar slants.

Spore generation

For spore generation, mycelia of P. palmivora were grown on V8 agar medium(50 g l�1 V8 juice, 0.2 g l�1 CaCO3 and 20 g l�1 agar) at 28 °C for four days. Sporangiawere induced by transferring two pieces of culture blocks (10� 10� 3mm) in 10mlsterile distilled water in Petri dishes for two days under white fluorescent light (Aragakiet al. 1967). Zoospores were released from sporangia by chilling at 5 °C for 30min.After filtration through muslin cloth, zoospore concentration was adjusted to 2� 103

spores per ml using a haemocytometer. Single drop of inoculum (20 μl; approximately200 zoospores) was used for infection studies.

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Plant material

A total of 10 cassava varieties were used in this study (Table 1).

Leaf inoculation

Healthy mature cassava leaves of each variety were collected and surface sterilised bywiping with 70% ethanol, then rinsed in sterile distilled water and blot dried. Prior toinfection, the midrib of the leaves was punctured with a needle. Single drop of inocu-lum was placed at the centre of the freshly made wound and was covered by wateragar. Leaves inoculated with water agar served as a control. The petiole of the leaveswas covered with wet cotton to avoid desiccation and was placed over a moist filterpaper in a plastic tray and incubated at 26–28 °C in the dark. Lesion development wasobserved for seven days. The lesion size was determined by measuring the length (A)and width (B) of the lesion and size calculated using the formula: (A +B)/2 (Kunkeawet al. 2010).

Whole tuber inoculation

Healthy tuberous roots from mature plants were collected, washed thoroughly underrunning tap water. They were surface sterilised by wiping with 1% sodium hypochlo-rite, washed in sterile distilled water and were briefly blot dried. The inoculation wascarried out as reported earlier (Senthil et al. 2012a). In brief, periderm tissue(5� 1mm) of the tuber was cut with a cork borer near the middle area of the tuber andinoculated by placing a single drop of inoculum (as described earlier) and was coveredby water agar. Control tubers were inoculated only with water agar. After inoculation,tubers were covered with two layers of wet paper towels to prevent dehydration andincubated in darkness at 26–28 °C for 10 days. To determine root rot severity, the

Table 1. Analysis of variance for leaves, tuber and stem of cassava cultivars in response toinoculation with P. palmivora.

Cultivars

Mean Duncan grouping⁄

Leaf⁄⁄ Whole tuber⁄⁄⁄ Stem cutting⁄⁄⁄⁄

H-165 2.46e �0.37c 15a

H-226 2.25f �0.37c 15a

Sree Harsha 3.0b �0.25b 15a

Sree Jaya 2.9c 0.14a 13.05d

Sree Padmanabha 0.80h �1.02e 15a

Sree Prabha 1.66g �0.64d 15a

Sree Prakash 2.7d 0.12a 14.24b

Sree Rekha 3.45a 0.11a 13d

Sree Sahya 2.43e �0.38c 14.06b

Sree Vijaya 3.06b �0.22b 13.66c

The results showed a significant correlation between leaf and stem with p value of 0.883. But negative corre-lation was for stem with leaf and tuber with the p value of �0.60 and �0.68.⁄Values are the mean of Duncan’s multiple range test of rot severity when tested for pathogenicity. Values fol-lowed by same letter do not differ significantly (p< 0.05).⁄⁄Leaf ratings are the average length and breadth of the lesion size.⁄⁄⁄Whole root ratings are the percentage of the mean severity (cm) of penetration of rot in infected tubers.⁄⁄⁄⁄Stem cutting ratings are the lesion length colonised by the pathogen in stem.

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maximum width (W) and depth (D) of rot from the inoculation point were measured,and the penetration of rot was calculated using the formula described by Lapwood(1967) i.e. (Penetration = (W/2 + [D – 5])/2).

Stem inoculation

Stem inoculation was performed as described by Onyeka et al. (2005b) with modifica-tions. In brief, cassava cuttings (15 cm long) were washed in running tap water for10min and surface sterilised in 1% sodium hypochlorite. The cuttings were blot driedon a sterile filter paper. Each cutting was inoculated at one end with single drop ofinoculum (as explained earlier) and was covered by water agar. The other end of thestem was covered with moist cotton. Stem cuttings inoculated with water agar served ascontrol. The cuttings were placed over a wet filter paper in a tray covered with a poly-thene cover and incubated at 26–28 °C for 10 days. Pathogen invasion was evaluatedafter peeling the bark of the cuttings by measuring the spread of the pathogen along thestem. The percentage length of the cutting colonised by the pathogen was recorded.

Statistical analysis

All the inoculation experiments were repeated thrice with three replicates. All data weresubjected to analysis of variance (SAS 2011), using the Statistical Analysis Systempackage. The Duncan multiple range test was used to identify the significant differences(p< 0.05) among the individual groups. The relationship between disease severity inleaf, stem and tuber was analysed with Pearson’s correlation test to determine thestrength of the correlation between these variables.

Results and discussion

The isolate recently isolated from infected sample or the isolate which was re-isolatedfrom the infected tuber by artificial means was used for analysis in order to ensure theaggressiveness of the isolate. The pathogen isolated exhibited white diffused mycelialgrowth on potato dextrose agar plates and spores were ellipsoid or oval with shortpedicel (Figure 1).

Zoospores were used for infection to stimulate the conditions identical in field. A100% disease incidence was observed in inoculated leaves, stem and tubers. Theinfected leaves exhibited black lesions and the infected tuber exhibited characteristicwatery brown discoloration. The disease severity varied between the varieties analysedand was grouped (Table 1). The disease severity was high on the cultivar Sree Rekhaand low on Sree Padmanabha showed its level of resistance. The cultivars grouped werealmost similar except Sree Prakash and Sree Harsha (Table 1). The infected stemexhibited brown lesion and lesion rate was almost similar (Table 1).

On correlation analysis, stem analysis showed poor correlation with leaf and tuber(�0.60 and �0.68) which shows that the mechanism of resistance is apparently inde-pendent and governed by different mechanisms. But the leaf and tuber results showedpositive correlation (0.88), providing the reliability of the analysis. The result suggeststhat the internal or tissue resistance to be common (Iwaro et al. 1997).

Of the results, stem-based analysis failed to discriminate the cultivars as did by leafand tuber analysis. The negative correlation showed by stem results is acceptable asresistance of different plant tissues to a pathogen may vary (Dhingra and Sinclair

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1986). Leaf-based analysis showed higher susceptibility when compared to whole tuberassay but was able to discriminate even minor level of variation in cultivars in responseto resistance reaction (see Figure 2).

The results from cassava leaf and tuber inoculation with P. palmivora revealed thatmost of the cassava cultivars grown are susceptible to rot disease. Iwaro et al. (2006)suggests that the high level of susceptibility among the cultivars could be due to theresult of progenies produced from susceptible parents. The susceptibility of cultivars toroot rot disease implies the lack and need of breeding programme in developingresistant cassava cultivars.

We believe that the selection of cultivars showing high resistance in both leaf andtuber analysis could be more valuable in breeding programmes or as markers. In presentstudy, the cultivar Sree Padmanabha showed resistance in both leaf and tuber analysis

Figure 2. Lesions caused by P. palmivora on tubers of cassava varieties: (a) Sree Padmanabha,(b) Sree Prakash, (c) Sree Vijaya and (d) Sree Rekha.

Figure 1. Colony morphology of P. palmivora on potato dextrose agar (a) and sporemorphology stained with lactophenol cotton blue under the light microscope. Scale bar represents10 μm (b and c).

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and further, it is claimed to be tolerant to cassava mosaic disease. The variety SreePadmanabha could play an important role in breeding programmes and could be apromising variety. Our result suggests that for resistant pre-screening, the leaf inocula-tion method could be a rapid and reliable method. The results of the present studies areof high importance, being the first on study in assessing the resistant level of cassavacultivars against root rot using in vitro tuber analysis. A further study on correlationwith field data is essential in boosting present findings.

AcknowledgementsThe funding provided for conducting the research work by the Indian Council of AgriculturalResearch, New Delhi, under IISR Outreach Programme is gratefully acknowledged. The authorsthank the Director for providing the infrastructure facilities and Dr J. Sreekumar, Senior Scientist,Central Tuber Crops Research Institute, Thiruvananthapuram, for his valuable support instatistical analysis.

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