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Changes in Foliar Glycoalkaloids Levels of Potato (Solanum tuberosum) Triggered by Late Blight Disease Severity.

Byline: Abdul Majeed Zubeda Chaudhry and Zahir Muhammad

AbstractGlycoalkaloids are secondary metabolites found in Solanum tuberosum L. and other members of Solanaceae which have potential role in the defense of host against certain fungi nematodes herbivores and other stress conditions. This study was undertaken to investigate the possible relationship between late blight disease severity and foliar glycoalkaloids of potato cv. Desiree. Disease severity and total glycoalkaloids contents determined after 3 6 9 and 12 days after inoculation with Phytophthora infestans showed no relationship when compared to control. Values of total glycoalkaloids contents of potato foliage with different late blight disease severity were almost consistent with control plants which were inoculated with sterile distilled water; although TGA levels in diseased plants showed slight but non-significant elevations than control.

Results indicated that disease severity had no effect on foliar TGA concentrations; however age of plant and length of inoculation period corresponded to higher glycoalkaloids contents and disease severity of leaves. Copyright 2014 Friends Science Publishers.

Keywords: Elicitors; Host defense; Phytoalexins; Phytophthora infestans; Secondary metabolites.

Introduction

Phytophthora infestans (Mont.) de Bary is perhaps the most widely studied plant pathogen which caused the Great Irish Potato Famine in 1840s by destroying potato crop-the then staple food of Irish people (Bourke 1993). P. infestans parasitizes a number of plants belonging to Solanacea (Turkensteen 1978) however it adversely affects potato and tomatoes by causing late blight disease (Tosun et al. 2007). Late blight of potato is still one of the major threats faced by potato growers throughout the world resulting in direct yield losses of crop in addition to huge monitory expenditures associated with fungicides application for controlling the disease (CIP 1997; Hijmans et al. 2000). Although late blight of potato can be controlled through rigorous fungicides applications

cultivating resistant varieties and integrated disease management strategies; modern research however focuses on inducing resistant traits in cultivated potato against various pathogens and pests (Lorito et al. 1998; Hoy 1999; Naqvi et al. 2012) through inter-specific hybridization and genetic transformation methods (Esposito et al. 2002). There is also dire need to identify the possible of role of secondary metabolites (phytoalexins) present in potato for anti- pathogenic activities. Understanding the relationship between the pathogens and secondary metabolites would be appealing for potato breeders to develop transgenic varieties with enhanced resistance to potential pathogens.

Glycoalkaloids are important secondary metabolites found in some genera of family Solanaceae including cultivated potato (Solanum tuberosum L.) in different concentrations at different plant parts (Osman 1983) which show toxicity to pathogenic fungi predators insects and pest (Tingey 1984; Matthews et al. 2005; Friedman 2006). Principle glycoalkaloids present in potato are a-chaconine and a-solanine (Freidman and McDonald 1997). Genetic factors age of plant stress conditions pathogenic attacks and herbivory are important determinants of glycoalkaloids levels in potato (Sinden et al. 1984; Friedman 2006). Their concentrations are usually higher in plant parts with high metabolism rate such as young leaves and flowers with subsequent decrease in parts with low metabolic activity upon maturity (Friedman and McDonald 1997).

Published reports on possible association between foliar/tuber glycoalkaloids of potato and late blight disease resistance are variable (Friedman 2006). Deahl et al. (1973) investigated the association of foliar and tuber glycoalkaloids and late blight disease resistance in fifteen potato clones however they reported lack of such association. Similarly Frank et al. (1975) reported no relationship between total glycoalkaloids of potato leaves and late blight disease resistance in field experiments. On the other hand Andreu et al. (2001) observed increased level of accumulation of glycoalkaloids and other phytoalexins in leaves and tubers tissues after inoculation with P. infestans but no correlation between the disease resistance and glycoalkaloids was established. The aim of this study was to investigate the effects of late blight disease on foliar glycoalkaloids levels and possible relationship between disease resistance and total glycoalkaloids contents of potato foliage.

Materials and Methods

Collection and Culturing of Phytophthora infestans

Culturing procedure for P. infestans was carried out at Plant and Environmental Protection unit National Agriculture Research Centre (NARC) Islamabad. Infected tissues of previously collected leaves were placed in petri-dishes containing rye agar medium supplied with antibiotic 100 L/mL vancomycin 100 L/mL pimaricin and 50 L/mL rifamycin (Caten and Jinks 1968). In order to promote sporulation petridishes were incubated for 4 days at 18C in dark. Freshly formed sporangia were transferred by sterilized glass rod to fresh rye agar medium without antibiotics and were re-incubated at 18C for 14 days in dark. Freshly formed sporangia were dislodged by sterile glass rod and adding 10 mL of distilled water to each petridish. For separating sporangia from mycelia fragments sporangial suspensions were filtered through a double layer of cheesecloth (pore size = 60 m; 14 x 12 threads centimeter-1) and concentrations were adjusted by haemo- cytometer to 60000 sporangia/mL. The sporangia were incubated at 4C for two h to induce germination (Mukalazi et al. 2001; Pliakhnevich and Ivaniuk 2008).

Determination of Foliar Glycoalkaloids

For determination of foliar glycoalakaloids Potato seed tubers (cv Desiree) were grown in four row plots 3 meter long with spacing of 70 cm between rows and 30 cm within rows at Botany Department Hazara University Mansehra in July 2011. Experiment was laid out in a randomized complete block design (RCBD) with four replications. Leaves were inoculated with 20 L sporangial suspensions at the center 45 days after planting. Control plants were inoculated with sterile distilled water. In order to prevent Phytophthora infection control plants were sprayed with contact fungicide Mandy Prompide (Revus) at three day interval. After inoculation disease severity (% leaflet infection) was calculated at 3 6 9 and 12 days after inoculation (Lebreton et al. 1999). Total glycoalkaloids of potato leaves (control and infected at different days of inoculation) were determined following the method of Dao and Friedman (1996) with minor modifications. Healthy and infected leaves were randomly collected from the middle parts of plants from control (water inoculated) and diseased plots (infected with P. infestans) at 0 3 6 9 and 12 after inoculation. A single large and fully expanded leaf (weighing 300 mg) was considered as a single experimental unit replicated four times for each treatment. Each leafsample was blended in 20 mL aqueous acetic acid (5%) to make a homogenate. The homogenate was then filtered through Whatman filter paper no. 4 and pH of the filtrate was maintained by ammonium hydroxide to 10. The filtrate from control and diseased leaves was used for total glycoalkaloids contents determination by HPLC technique at PCSIR Laboratory complex Peshawar using HPLC Shimadzu SC 6A System. For reducing biasedness of results the procedure was repeated four times.One way ANOVA was used to analyze disease severity while for finding association between means of glycoalkaloids and disease severity two-way ANOVA was applied to collected data. Means were separated by LSD at p= 0.05.

Results

The objective of this study was to investigate the relationship between late blight disease severity and total glycoalkaloids of potato leaves. Results indicated that days after inoculation (DAI) had significant effect on the disease severity (Table 1). At 3rd DAI disease severity was recorded as 5.263% with increasing tendency as DAI progressed; 12th DAI revealed maximum disease severity 70.135 % (Fig. 1).In order to determine the effect of disease severity on glycoalkaloids foliar total glycoalkaloids of both diseased plants and control were determined at 3 6 9 and 12th day of inoculation. Foliar TGA values of diseased plants were compared with control at each assessment period. Results showed that TGA determined before inoculation of leaves with late blight pathogen and sterile distilled water (in control) was 29.43 mg/100 g fresh weight (Fig. 2). TGA contents increased significantly with age of plant. Increase in TGA levels in both control and diseased plants were almost consistent although diseased plants showed slightly higher values of TGA than control but these results were insignificant. TGA determined after three days of inoculation in diseased plants were 43.08 mg/100 g fresh weight whereas in control (inoculated with sterile distilled water) plants 42.14 mg/100 g fr. wt. of TGA were recorded. Likewise slight but insignificant increase in glycoalkaloids levels was also recorded at 6 9 and 12 DAI (50.80 52.81 and 75.67 mg/100 g fr. wt.) when compared to control where TGA values were 49.33 52.18 and 74.28 mg/100 gfr. wt. respectively (Fig. 2). ANOVA did not reveal any correlation between disease severity and TGA of leaves determined at different assessment periods however impact of days after inoculation (DAI) were significant on glycoalkaloids levels in both control and diseased plants (Table 3). At 3rd day of inoculation foliar TGA in diseased plants increased by 2.230% than control. Maximum increase (2.970%) was recorded at 6th DAI followed by 9th DAI (1.207%) and 12th DAI (1.870%) respectively (Table 2). Contrarily TGA levels in both diseased and control plants significantly increased with the increase in days after inoculation (DAI) i.e. TGA levels were higher in both diseased and control at 12 DAI followed by 9 DAI 6 DAI and 3 DAI respectively (Fig. 2). Comparing TGA levels of diseased plants with control at each DAI result clearly demonstrated that disease severity had no effect on the TGA levels. On the other hand TGA levels in diseased and control plants were significantly increased by increase in DAI i.e. age of the plants (Table 3).

Discussion

Previously Deahl et al. (1973) and Frank et al. (1975) investigated the effect of late blight disease and some other pathogenic fungi on total glycoalkaloids of different potato clones but were unable to find association between late blight disease and leaf TGA contents. Andreu et al. (2001) reported that glycoalkaloids phenolic compounds and phytoalexins slightly but insignificantly increased in potato leaves after inoculation with P. infestans; however association between disease severity and glycoalkloids could not be established in their studies.

Glycoalkaloids are important secondary metabolites of members of family Solanacea including potato. Principle glycoalkaloids present in potato are a-chaconine and a- solanine (Freidman and McDonald 1997). In potatoes like other Solanacea members concentrations of these compounds are dependent on several factors like age of plant climate varieties biotic and abiotic stresses (Sinden et al. 1984; Friedman 2006; Khan et al. 2013). Young tissues of plant have maximum levels of glycoalkaloids

Table 1: ANOVA table of mean square for disease severity (%)

Source###Degrees of freedom###Sum of squares###Mean square###F-value###Prob.

Replications###3###0.01###0.003###4.95###0.0268

Treatment###3###9795.06###3265.020###5966131.65###0.0000

Error###9###0.00###0.001

Total###15###9795.07

Table 2: Effect of disease severity on foliar total glycoalkaloids of potato determined at different inoculation periods.

Values in parenthesis in the third column represent percent increase in TGA to control

Disease severity (%)###Days after inoculation (DAI)###Foliar TGA (mg 100 g-1 fresh wt.)###LSD value at p=0.05

###Inoculated with P. infestans Control (inoculation with distilled water)

0###0###29.43a###29.43a###-

5.263###3###43.08b (2.230)###42.14b###1.204

23.14###6###50.8c (2.970)###49.33c###2.86

49.308###9###52.81c (1.207)###52.18c###0.987

70.135###12###75.67d 1.870)###74.28d###2.023

LSD value at p=0.05###5.129###4.71###-

Table 3: Effect of days after inoculation (DAI) on Total Glycoalkaloids (TGA) of potato leaves

K value###Source###Degrees of freedom###Sum of squares###Mean squares###F value###Prob

1###Replications###3###1.858###0.619###0.9438

2###TGA###1###1.069###1.069###1.6292###0.212ns

4###DAI###4###8859.782###2214.945###3374.7968###0.0000

6###TGA x DAI###4###9.690###2.423###3.6911###0.0160

-7###error###27###17.721###0.656

###Total###39###8890.120

concentrations which increase with plant maturity and become even higher during flowering stage (Friedman2006). Similarly environmental stresses and pathogenic attacks may also contribute to variation in glycoalkaloids levels indicating their possible role in defense of host and disease resistance (Andreu et al. 2001).The mechanism of the potential role of glycoalkaloids in disease resistance and host defense is not well understood. However it is assumed that changes in glycoalkaloids or other phytoalexins after infection by a pathogen is triggered by either chemicals released from the pathogen or it may be due to the host and pathogen interaction. Defense response of the host to the pathogenic attack results in the production or induction of changes (increase or decrease) in phytoalexins which may correlate with the pathogen in a positive or negative way (Hammerschmidt 1999). The defense response of the host is generally initiated by elicitors- molecules released by the pathogen or produced by the host in response to pathogen interaction (Hammerschmidt 1999; Sharma et al. 2011). In turn the host may possibly produce toxins resistant to the pathogen's growth and feeding; or may trigger other metabolites and enzymes of the host for a prompt response to the stress caused by the pathogen. The antifungal and pesticidal activity of glycoalkaloids particularly a-solanine and a-chaconine are presumed to be because of their ability to bind with and disrupt cell membranes of the pathogens (fungi insects pest) having high sterols (Martin and Douglas 1997). Sterol binding destabilization of cell membranes and inhibition of enzymes by glycoalkaloids in different studies have been confirmed (Roddick et al. 19882001). Differences in sensitivity of glycoalkloids of potato to different pathogens may possibly be because of differential concentrations of cell membrane's sterol contents in different pathogens (Martin and Douglas 1997). Thus one of the possible answers for the lack of correlation between glycoalkloids of potato and late blight disease caused by P. infestans as indicated in many studies may probably be due to low sterols contents in cell membrane of P. infestans; hence low binding and membrane disruption capacity of these compounds with P. infestans (Nes et al.1983; Roddick 1987).In conclusion foliar total glycoalkaloids contents of potato studied at different timing after inoculation with Phytophthora infestans did not varied significantly than foliar TGA contents of control plants and thus no correlation between disease severity levels and glycoalkaloids contents of potato leaves could be established in our study. However TGA contents in both control and test plants significantly increased at different inoculation periods revealing that these compounds are dependent on the age of plants.

Acknowledgements

First author extends thanks to Higher EducationCommission (HEC) Government of Pakistan for financing his PhD studies through 5000 Indigenous PhD Fellowship Program (Batch IV). Authors are also thankful to Dr. Mendel Friedman (Agricultural Research Service U.S. Department of Agriculture) for providing his important papers on potato glycoalkaloids.

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Publication:International Journal of Agriculture and Biology
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