Virulence of Phytophthora isolates from Piper nigrum L. and their sensitivity to metalaxyl-mancozeb

Foot rot disease caused by Phytophthora capsici is the most destructive disease in black pepper. A total of 82 isolates from the National Repository of Phytophthora at ICAR-Indian Institute of Spices Research were evaluated for their sensitivity to metalaxyl-mancozeb and also tested for their virulence. Of the 82 isolates studied, 19 (23.2%) were highly sensitive to metalaxyl-mancozeb and 6 (7.3%) were comparatively insensitive (EC 90 value >40 ppm; range 41.3 to 68.5 ppm). The isolates also varied in the degree of virulence, viz. , 34 (41.5%) were highly virulent, 39 (47.6%) moderately virulent, 4 (3.7%) mildly virulent, and 5 (6.1%) non-virulent. There was neither any significant correlation between sensitivity to metalaxyl-mancozeb and virulence nor with the geographical location.


Introduction
Phytophthora foot rot, caused by Phytophthora capsici, is a serious threat to the production of black pepper (Piper nigrum) worldwide (Anandaraj, 2000). This oomycete is soil-borne, but also spreads through splashing of raindrops and infects roots, stem, leaves, and fruit at any stage of plant growth. Infection on plant parts below the ground cannot be detected during the initial stages of infection. Symptoms appear only at the advanced stages of root rot, and measures to control the disease at this juncture are futile. Preventive application of fungicides such as Bordeaux mixture, copper oxychloride, metalaxyl-mancozeb, or potassium phosphonate (Anandaraj and Sarma, 1995) to the soil or foliage is the recommended practice. A phenyl amide fungicide, namely metalaxyl, was found to be highly effective (Daggett et al., 1993) but its high efficacy and specificity prompted its widespread use, which led to the emergence of P. infestans isolates resistant to the fungicide. Such resistance was first reported in the mid-1980s in P. infestans from the Netherlands and Ireland (Davidse et al., 1981a(Davidse et al., , 1981bDowley and O'Sullivan, 1981;Gisi et al., 1985), followed by resistance to metalaxyl in Korea (Choi et al., 1992 andKim et al., 2003). To counter such resistance, mixtures of fungicides are preferred and currently used for managing the diseases caused by Phytophthora. Such mixtures help in three ways, by (1) widening the spectrum of activity; (2) exploiting synergy between fungicides, which increases the overall impact and also permits lower doses of individual fungicides; and (3) delaying the emergence of resistant isolates. Ramachandran (1990) studied the sensitivity of P. capsici isolates of black pepper to metalaxyl, and Veena et al. (2010) found a differential response in 29 isolates of P. capsici to potassium phosphonate in black pepper. Given the wide morphological diversity among Phytophthora strains isolated from different blackpepper-growing tracts as well as from different microclimates (the rhizosphere and different plant parts), it is imperative to study the isolates for variation in virulence and sensitivity to metalaxyl. The present work widened the scope of that objective by looking for any correlation between these attributes and the location from which the isolates had been collected and also between sensitivity to the fungicide and virulence.

Phytophthora isolates
Eighty-two isolates of Phytophthora, representing all the three major states in India in which black pepper is grown, were chosen for the study: 44 from Kerala, 36 from Karnataka, and 2 from Tamil Nadu (Table 1). These isolates form part of the National Repository of Phytophthora maintained at the ICAR-Indian Institute of Spices Research (IISR), Kozhikode, Kerala. The isolates had been collected over time (1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010) from roots, stems, leaves, and fruits (berries) of black pepper and also from the collar region and the rhizosphere. The isolates maintained in sterile water were purified using PVPH medium (Tsao and Guy, 1977) and sub-cultured onto carrot agar (CA) when required.

Metalaxyl sensitivity
Sensitivity to the fungicide metalaxylmancozeb was determined by growing the isolates on CA supplemented with a commercially available formulation of the fungicide mixture (available as a wettable powder, containing eight per cent metalaxyl and 64 per cent mancozeb).
A preliminary study indicated that there was complete inhibition at 49.5 ppm and hence, further lower concentrations were used in this study. Stock solution of the fungicide (200 ppm) was prepared in sterile distilled water. Appropriate quantities of the stock solution were incorporated into sterilized CA, which was cooled to 49 °C before mixing, to obtain the following concentrations: 0.9, 4.5, 9.0, 13.5,18, 22.5, 27, 36, 45, and 49.5 ppm (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, and 5.5 ppm of metalaxyl respectively) (Parra and Ristaino, 2001;Silvar et al., 2006). Agar plugs (5 mm in diameter) cut from the growing margins of 72-hour-old cultures of the isolates were placed at the centre of the plates containing different concentrations of the fungicide and incubated at 25 °C for 72 h. Control plates (without the fungicide) were subjected to the same procedure. The experiment was laid out as a threefactor completely randomized design with three replicates, and each concentration was replicated three times. The colony diameter was measured at 72 h and percentage inhibition (PI) was calculated as follows: PI = [C -T /C]×100, where, C is the measure of growth of the pathogen in control plate (mm) and T is the measure of growth of the pathogen in treatments.
Based on the PI, the effective concentrations (EC 50 and EC 90 ) were calculated by probit analysis using SPSS (ver. 19) and the isolates were scored as sensitive (S), intermediate (I), or insensitive/ less sensitive (L), depending on the concentration (ppm) of metalaxyl-mancozeb in CA viz., S -EC 90 at less than 10 ppm; I -EC 90 at 10 to 40 ppm; L -EC 90 at more than 40 ppm. Furthermore, values of EC 50 for each isolate were calculated by plotting the percentage of growth of each isolate against log10 of the metalaxyl-mancozeb concentration.

Virulence assay
All the isolates were tested for their virulence on black pepper based on the detached leaf assay (Bhai et al., 2010;Forbes, 1997). Leaves from threemonth-old rooted cuttings of Subhakara, a variety of black pepper susceptible to Phytophthora, which were raised in a greenhouse were used for the assay. The second leaf from top was excised, washed thoroughly with sterile water, and blotted dry with a sterile blotting paper. Mycelial plugs, 5 mm in diameter and cut from the growing margins of 72-hour-old cultures of each isolate were placed on the abaxial surface of the leaves and kept in closed plastic boxes lined with moistened filter paper. A small cotton pad moistened with sterile water was placed on the culture disc to keep the disc moist. The boxes were then incubated at 24±1 ºC. Lesions diameter was recorded every 24 h for 72 h and the diameter recorded at 72 h was used for scoring the virulence of the isolates on a scale of 1 to 4 as follows: 1 = no lesion (non-virulent), 2 = 1-10 mm (less virulent), 3 = 10.1-30 mm (moderately virulent), and 4 = more than 30 mm (highly virulent).
The data were analysed using the general linear model (GLM) procedure of SAS ver. 9.3 (SAS Institute, Cary, North Carolina) and the mean value comparisons were separated using Fischer's least significant difference (LSD) test. As some of the values were zero, square-root transformations (x + 0.5) were used for normalizing the variance. The entire experiment was repeated twice, and the data were pooled for statistical analysis. Significance was defined at p<0.05.

Sensitivity to metalaxyl
The in vitro studies revealed that the isolates were highly variable in terms of their sensitivity to metalaxyl-mancozeb where the EC50 value ranged from 0.0001 ppm to 14.31 ppm. The lowest EC50 was shown by an isolate (Accession No. 98-156) from Sirsi, Karnataka. Values of EC90 ranged from 0.49 ppm to 68.50 ppm. Among the 82 isolates, 19 (23.2%) were highly sensitive (EC90 0.49-9.88 ppm). Six isolates (7.3%) were the least sensitive (EC90 41.31-68.50 ppm at concentrations below 40 ppm) (Table 2, Fig. 1 and Fig. 2). A majority of the isolates (69.5%) were in the intermediate range.
However, these sensitive isolates showed no correlation between sensitivity and location of isolation.
Although only a few isolates proved resistant to metalaxyl-mancozeb in the present experiment as there is no wide spread use of metalaxyl in India for black pepper. Whereas, in the tropical highlands of Africa where potatoes are grown, it was reported that 86 per cent of P. infestans isolates from Kenya and 59 per cent from Uganda were resistant (Erselius et al., 1999). In Uganda, 44 per cent of P. infestans isolates were reported to be resistant to  (Mukalazi et al., 2001). High frequencies of metalaxyl resistance in P. infestans have also been reported elsewhere (Cohen and Coffey, 1986;Davidse et al., 1981b;Deahl et al., 1993;Gisiand Cohen, 1996;McLeod et al., 2001;Vega-Sanchez et al., 2000). Thind et al. (2001) reported variation in sensitivity to metalaxyl in P. infestans in Punjab, India. Out of the 68 populations studied, three were identified as resistant to metalaxyl. Fontem et al. (2005) studied the pathogenicity and metalaxyl sensitivity of P. infestans isolates obtained from garden huckleberry, potato, and tomato in Cameroon and found that the aggressiveness of the isolates increased with their resistance to metalaxyl. metalaxyl, and any alteration in the target site can lead to resistance in some pathogenic oomycetes.
From the genetic studies of P. infestans and P. sojae, Bhat et al. (1993) and Lee et al. (1999) reported that resistance was linked with a single dominant gene, and variations in resistance were accounted for by the influence of minor genes. Fabritius (1997) believed that metalaxyl resistance in P. infestans developed independently in different areas of the world.

Virulence assay
Among the total isolates, only five (6.1%) were non-virulent (they failed to induce any lesion); four (3.7%) proved to be less virulent (lesion diameter 2.6-3.6 mm); and the rest were either moderately virulent (47.6%) or highly virulent (41.5%; lesion  Dunn et al. (2010) studied the population structure and resistance to mefenoxam of P. capsici in New York State and found that the population to be highly diverse, but the gene flow among regions and fields to be restricted. Therefore, they concluded that the population from each field needs to be considered as independent population, and the movement of inoculum across the fields restricted to prevent the spread of the pathogen. Overall, worldwide populations of P. capsici vary in their sensitivity to fungicides and in virulence. Although only very few of the Phytophthora isolates of black pepper were resistant to metalaxyl, it is important to consider segregation as one of the disease management strategies. Our results are also very well supported by the work of Silvar et al. (2006), who characterized P. capsici isolates obtained from pepper (Capsicum annuum L.) in North-western Spain based on virulence, mating type, sensitivity to metalaxyl, and genetic analysis using random amplified polymorphic DNA methods and found no correlation between the categorization based on RAPD analysis and that based on virulence or response to metalaxyl. Davidse et al. (1983) reported RNA polymerase as the target site for diameter 17.0-28.3 mm) (Fig. 3). All the highly virulent isolates, 69.2% of the moderately virulent isolates, and only one among the less virulent isolates produced signs of infection on the leaves within 24 h of inoculation whereas the rest took 48 h or more to do so (data not shown). Though the isolates varied in the degree of aggressiveness in infecting the susceptible black pepper variety, neither virulence nor sensitivity to metalaxyl-mancozeb could be correlated with the location (origin) from which the isolates had been collected. Among the 82 isolates, only five were non-virulent but the age of the culture had nothing to do with the degree of virulence. The isolates collected in 1997 (97-52) were highly virulent whereas those collected in 2009 (09-25) were nonvirulent. Hwang et al. (1996) reported similar results in their studies on the variation in virulence in naturally occurring isolates of P. capsici. Variation in virulence among isolates of P. cactorum and P. parasitica was reported by Hantulaet al. (1997), Lebretonand Adrivon (1998, and Matheron and Matejka (1990). Similarly, varying levels of disease were observed with nine isolates of P. capsici on nine Korean and Japanese pumpkin cultivars (Lee et al., 2001), thus showing the difference in virulence among isolates. The diversity in virulence observed among P. capsici isolates of black pepper in this study is well supported by these studies. The sensitivity to metalaxyl-mancozeb and virulence were independent attributes. Out of 82 isolates studied, 41.5 per cent of the isolates were highly virulent but only 7.3 per cent were resistant to metalaxyl-mancozeb.

Conclusions
Only a small proportion (7.3%) of the isolates was resistant to metalaxyl-mancozeb, suggesting that the chance of developing resistance by the pathogen for this combination is negligible. Unlike vegetables in general and potato in particular the use of metalaxyl to manage Phytophthora infection is very negligible. That could be one of the reasons for the low frequency of resistant isolates encountered among black pepper isolates. The highly sensitive isolate from Sirsi 98-156 is from soil and never exposed to metalaxyl fungicide also suggests this. The farmers in this location manage Phytophthora by use of copper fungicides only as aerial spray. The variable sensitivity must be the intrinsic character of each isolate and not due to their exposure to the fungicide. There was no significant correlation between metalaxyl sensitivity and virulence of the isolates. Further studies are warranted to determine the molecular basis of this variation in fungicide sensitivity. Such studies can also help to identify markers linked to virulence/avirulence loci in Phytophthora of black pepper.