OCCURRENCE OF DAMPING OFF OF CHILI CAUSED BY PYTHIUM SPP. IN THE POTHWAR REGION OF PAKISTAN

The per acre yield of chili in Pakistan is quite low as compared to large producers of this vegetable in the world and can be attributed to an assortment of factors both biotic as well as abiotic. Among biotic factors, chili is susceptible to damping off caused by Pythium spp . and causes lots of damage in Pakistan. There is scantly information on the incidence and severity of Pythium associated with chili. Therefore, the current studies were carried out to determine the incidence and severity of Pythium in the Pothwar region of Pakistan. An overall incidence of 14.08% of Pythium damping off was recorded on chili in Pothwar. Attock showed the maximum incidence of 19.86% followed by Rawalpindi and Chakwal districts having 16.22% and 10.22% disease incidences respectively. Jhelum had the minimum disease incidence of 10.05%. Disease severity was the maximum in district Attock (11.03%) followed by Rawalpindi (9.11%), and was the minimum in Chakwal (5.42%) followed by Jhelum (5.67%). The finding of these studies will be helpful for the farmers in designing control strategies for this pathogen accordingly.


INTRODUCTION
Chili (Capsicum annuum L.) enjoys an important position in the world and is considered among the most favorite vegetables after potato and tomato. The vegetable belongs to the family Solanaceae (Berke, 2002). Chili is one of the most common condiments used in Pakistan and other South Asian countries. Chili is rich in vitamins, especially pro-vitamin A, B1 (thiamine), B2 (riboflavin), B3 (niacine), vitamins C, E, P (citrin). It also contains appreciable amounts of Phosphorus, Calcium, and Iron (MacGillivary, 1961;Macrae et al., 1993;Bosland and Votova, 1999). Chili is an herbaceous plant and is extremely sensitive to cold (Wein, 1999). It requires hot environment for its optimal growth and flourishes well on well-drained, silt loam or sandy soils. Chili plants have fibrous taproots which extend vertically 30-60 cm deep and horizontally 30-50 cm. Flowering occurs three months after planting. Flowers are hypogynous and hermaphrodite. Chili crop matures after 4 months of planting in dry and hot weathers. Chili can be cultivated under a wide range of environmental conditions; both subtropical and tropical but in general it requires temperature of 24-30 °C. In Pakistan, chili nursery is prepared during August for autumn/winter crop and in February to April for summer crop. In hilly areas, nursery is prepared in March to April. Thirty to fifty days old seedlings from the nursery are transplanted into the fields. Fully grown fresh green chilies are used as vegetable and for pickles. Red fully ripe chilies are picked for dried chili powder. The per acre yield of chili in Pakistan is quite low as compared to large producers of this vegetable in the world and can be attributed to an assortment of factors both biotic as well as abiotic (Ahmad et al., 2017;Asghar et al., 2020;Shahbaz et al., 2015). Chili becomes more susceptible to a wide range of fungal, bacterial, nematodal and viral diseases under natural environmental conditions of South East Asia (Ashfaq et al., 2014;Tazeem et al., 2018;Tariq-Khan et al., 2017;Tariq-Khan et al., 2020a;Green, 1991;Hameed et al., 1995;Tariq-Khan et al., 2020b). Fungi and bacteria cause foliar (leaf), fruit, stem or root diseases (Kamal and Mughal, 1968;Hafiz, 1986;Majid et al., 1992;Mushtaq and Hashmi, 1997;Aslam et al., 2017). For farmers, these diseases cause considerable yield losses which result in huge economic loss. Among fungal diseases, damping off caused by Pythium spp., late blight caused by Phytophthora spp. (Hyder et al., 2020) and Fusarium wilt are some of the most important diseases causing lots of damage to tomato and chili in Pakistan. Pythium damping off is a major nursery problem in chili fields (Sherf and MacNab, 1986;Alhussaen et al., 2011) where the organism kills newly emerged seedlings (Jarvis and Jarvis, 1992;Agrios, 2005;Redekar et al., 2019). Pythium spp. have a broad host range and causes pre-emergence and post-emergence damping-off and root rots followed by considerable yield losses in chili crop. Pythium species are rarely host specific (Rangaswamy, 1989). Pythium, a soil borne oomycete pathogen is "fungal-like" organism and a member of the family Pythiaceae. The fungus is able to spread from diseased seedlings to healthy plants through zoospores. Pythium species are homothallic; sexual reproduction can occur between the same hypha when the female oogonium and male antheridium fuse together as a result their nuclei unite to form a single zygote. At high temperatures (28 °C), the oospores or zygote germinate and develop into mycelium. Infectious zoospores develop at lower temperatures of 10 to 17 °C (Alexopoulos et al., 1996). There are a number of soil inhabiting pathogenic Pythium spp. i.e. P. aphanidermatum, P. ultimum, P. helicoides, P. myriotylum, and P. splendens which can cause significant root rot and reductions in root growth of chili crop. P. aphanidermatum can cause severe root rot, reductions in plant weight and 42-62% plant mortality under conducive conditions in chili plants. There are some other Pythium spp. which cause low incidences of root tip necrosis in chili plants. Root tip necrosis causing Pythium spp. are P. arrhenomanes, P. catenulatum, P. graminicola, and P. irregulare, but none of these species cause losses in root weight and only P. irregulare reduced shoot weight. The colonization of chili plants by P. aphanidermatum was observed to be affected by temperature. At 28 °C, reductions in growth occurred while at 34 °C plant mortality occurred in chili transplants (Chellemi et al., 2000). There is scantly information on the incidence and severity of Pythium species infecting chili in Pakistan especially in the Pothwar region. Therefore, the present studies were carried out to determine the incidence and prevalence of Pythium in the Pothwar region of Pakistan. The findings of these studies will be helpful for the farmers in designing control strategies for this pathogen accordingly.

MATERIALS AND METHODS
The studies reported in this paper were carried out in the Pothwar region of Pakistan and in the Department of Plant Pathology of Pir Mehr Ali Shah Arid Agriculture University Rawalpindi during 2017-2020.

Survey for incidence and severity
Chili fields of the Pothwar Plateau ( Figure 1) were surveyed to determine the incidence and severity of Pythium spp. during 2017-2020. In order to determine the incidence and severity, 15 sites were selected randomly from each district and from each site, 3 fields were selected. From each field, 50 plants were selected each after every 5 steps across a diagonal following hierarchical sampling strategy (McDonald and Martinez, 1990). Incidence of Pythium in each field of each site was calculated by the following formula; ( ) The incidences of three fields were averaged to determine the incidence of each site. Similarly, the incidences of 15 sites of each district were averaged for determining the incidence of that district. Individual diseased plants of chili from each field of each site were assessed for severity of Pythium by following severity index scale developed by Altier and Thies (1995) (Table 1).

Collection of samples
Five diseased infected plants of chilli were randomly selected on the basis of symptoms from each field following the hierarchical sampling strategy (McDonald and Martinez, 1990). For the isolation of Pythium, soil samples were taken from the rhizospheres of randomly selected plants at the soil depth of 12 cm. Samples of each field were mixed to obtain pooled sample. The collected samples were labeled properly and immediately brought to the laboratory to start isolation of Pythium species. Soil samples containing roots of Pythium infected chili plants were placed in plastic bags and thoroughly moistened with autoclaved water. Potato plugs of 10 mm diameter and 50 mm lengths were added into each bag. The potato tubers were washed, peeled and surfacedisinfected before cutting into plugs with a sterile cork borer. After 24 hours of incubation at room temperature, the plugs were rinsed with tap water and cut into small lengthwise slices and plated on to Pythium V8 media amended with antibiotics.

Purification of cultures
Pure cultures of the fungal pathogens were obtained by single spore and hyphal tip method. In petriplates where Pythium have produced zoosporangia, pure cultures were obtained using single spore method, by picking a single zoosoprangium of fungus with the help of sterilized needle under stereoscope placed in laminar air flow hood and was transferred to petriplates and slants containing selective media. The plates and slants were incubated at 25 °C. In petriplates where Pythium have not produced zoospores, pure cultures were made by hyphal tip method. To obtain pure culture, growing hyphal tips were cut along with media (using cork borer) and were placed onto the surface of petriplates containing fresh selective medium. The pletriplates were incubated for 15-24 hours at room temperature.

Identification of Pythium species on the basis of morphology
Water cultures of Pythium were prepared. Inoculum discs were added to sterile water in petriplates along with chili leaves and were incubated at 25 ℃. Pythium species were identified based on structure of sporangia, zoospore and sexual structures. Pythium isolates were identified morphologically as described by Van der Plaats-Niterink (1981) and Dick (1990). The purified and identified cultures of all the isolates of Pythium were maintained at 25 °C.

Pathogenicity bioassay
Chili seeds were grown in trays in the greenhouse. Four to six leaf seedlings of chili were inoculated with the already prepared inoculum. The roots of seedlings were inoculated by drenching method. Plants were covered with a plastic sheet for 16 hours to prevent the inoculum from drying. Symptoms were observed and recorded.

Incidence and severity of Pythium spp. damping off on chili in Pothwar
Pythium damping off showed an overall incidence of 14.08% in the Pothwar region. Attock showed the maximum incidence of 19.86% followed by Rawalpindi and Chakwal districts having 16.22% and 10.22% disease incidences respectively. Jhelum had the minimum disease incidence of 10.05% as shown in Figure 2. Disease severity was the maximum in district Attock (11.03%) followed by Rawalpindi (9.11%), and was the minimum in Chakwal (5.42%) followed by Jhelum (5.67%) as shown in Figure 3.          In district Chakwal, the maximum incidence of 11.33% was recorded in Mureed and Chakral. On the other hand, Norwal area had the minimum incidence of 4.00%. In the same way, Pinwal area had the maximum disease severity of 8.10% while Norwal area showed the minimum disease severity of 2.00%. The severity and incidence of Pythium damping off on chili crop in each locality of district Chakwal are given in Table 4. As regards district Jhelum, the maximum incidence was observed in Kala Gujran (16.66%) while it was the minimum in Jatipur (7.33%). Kala Gujran and Jatipur also showed the maximum and the minimum disease severity values of 9.20% and 4.00% respectively. Disease incidences and disease severities in various areas in district Jhelum are given in Table 5.

Morphological characterization of Pythium spp.
In total, 39 isolates were recovered from chili crop in the Pothwar region. All the recovered isolates were morphologically characterized using different parameters like colony color, growth habit, hyphal swellings, size and shape of sporangia, cardinal temperature, sexuality, oogonia and anthredia. For microscopic identification, fresh slides were prepared from pure cultures of each isolate and were visualized under high power microscope. Thirty of the recovered isolates produced loosely to compact aerial mycelium with no distinct pattern, hyphae were aseptate, hyphal swellings were absent and sporangiophores were filamentous. Isolates also responded similarly to critical temperature range (Min: 10±1 °C, Max: 40±1 °C, Opt: 28±1 °C). All the isolates were morphologically confirmed to be Pythium aphanidermatum as already done by various scientists. Rest of the 9 isolates showed variable morphology from colony color of hyaline, grayish with loosely to compact aerial mycelium with no distinct pattern; mycelium was aseptate, with distinct hyphal swellings and sporangia were globose. Oogoina were terminal and anthredia were monolicious. These 9 isolates were confirmed to be Pythium ultimum.

DISCUSSION
Pythium is a renowned soil borne Oomycete pathogen with a wide host range in warmer parts of the world. Pythium damping off is a very important and common problem in chili and tomato fields (Redekar et al., 2019;Agrios, 2005;Alhussaen et al., 2011;Sherf and MacNab, 1986), where the organism kills newly emerged seedlings (Redekar et al., 2019;Jarvis and Jarvis, 1992). Pythium species are rarely host specific (Rangaswamy, 1989). In the present study, incidence and severity of Pythium damping off on chili was observed. Incidence and severity of Pythium damping off on chili was the maximum in Rawalpindi (16% and 9%) and Attock (19% and11%) and the minimum in Chakwal (10% and 5%) and Jhelum (10% and 5%). This high incidence of Pythium in Rawalpindi and Attock can be attributed to different factors. Pythium is a soil borne pathogen. Its spores can survive long, harsh periods as resting spores and also have the ability to travel through water. Rawalpindi and Attock have a relatively high humid climate as compared to Chakwal and Jhelum which usually have low rainfall. High moisture contents in the soil support the motility of zoospores and increase the size of the spermosphere (Martin and Loper, 1999;Martin, 2000). It has been observed that high moisture contents of soil results in increased disease incidence and severity of Pythium damping-off on chili crop (Muthukumar et al., 2009;Hyder et al., 2018a;Hyder et al., 2018b;Majeed et al., 2019). Greenhouse condition of high moisture and high temperatures provides thriving conditions for Pythium (Kerkeni et al., 2007;Manjunath et al., 2010;Arora et al., 2021). It is well documented that Pythium colonization of chili and tomato plants was observed to be affected by temperature. Although chili and tomato plants can survive at high temperatures but when plants are infected by Pythium spp. growth of chili is reduced at 28 °C while at 34 °C plant dies (Chellemi et al., 2000). Farmers in Rawalpindi and Attock areas also grow other solanaceous crops e.g. potato and tomato. As it is known that Pythium has a wide host range and are less host specific and continuous availability of other hosts increases their survival in soil (Rangaswamy, 1989). Furthermore, the farmers are incognizant about the mechanism of spread of this disease and preventive measures are not taken. Due to this main reason, the spread of the disease is increasing in the Pothwar region.

CONCLUSION
It is concluded from the present studies that Pythium damping off is fairly distributed in the Phothwar region of the Punjab province of Pakistan and warrant stringent control strategies for its management. Further, the findings of these studies will be helpful for the farmers in designing control strategies for this pathogen accordingly. The information will also be useful for the breeders to develop resistant varieties of chili against the most virulent isolates of the pathogen.