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Morpho-physiological Studies Management and Screening of Tomato Germplasm against Alternaria solani the Causal Agent of Tomato Early Blight.

Byline: Sobia Chohan Rashida Perveen Mirza Abid Mehmood Safina Naz and Naila Akram

Abstract

Early blight (Alternaria solani) is a potential disease of tomato that reduces its production globally both in conventional and tunnel cultivations. Due to variability in pathogenic isolates prolonged active disease cycle phase and broad host range early blight is very difficult to manage. A. solani isolate collected from tunnel grown under vegetable area Bahauddin Zakariya University Multan was subjected to different temperature range pH levels light intensity and growth media (in vitro). The results indicated that A. solani grew the maximum at 25C (7.50 cm) on PDA medium at 6.5 pH (8.34 cm) under continuous light condition (9.00 cm). On PDA medium the pigmentation varied from creamy yellow brown black to olivacious brown while on HLEA medium it was light brown. Varietal screening of six tomato varieties was carried out against early blight disease. No variety was found resistant.

Concerning severity out of six tomato varieties Roma showed maximum susceptibility (70.50%) while in Nagina it was least (29.38%). Efficacy of three fungicides (Topsin M Bavistin and Ridomil Gold MZ) at 1 g/L 2 g/L and 3 g/L concentration and two bio-agents (Trichoderma harzianum and T. viridae) was evaluated against A. solani in vitro and under tunnel cultivation. In in vitro assay Ridomil Gold MZ inhibited A. solani (47.06%) at 2 g/L while at 3 g/L concentration Topsin M was more effective (64.71%) as compared to control. Comparing both bioagents inhibition percentage (67.78%) was recorded in T. harzianum whereas T. viridae showed less inhibition (59.63%). Under tunnel cultivation early blight of tomato was significantly reduced by foliar applications of Topsin M and T. harzianium at 3 g/L and 108 conidia/mL concentration respectively comparing with untreated plants.

In the light of present study farmers could be suggested the practice of resistance source combination of management practices and avoidance of environmental conditions favoring the pathogen thus result in significant production of tomato.

Keywords: Bavistin; Conidiophores; Light regimes; Roma; Topsin M; Trichoderma Species

Introduction

Solanaceae commonly known as nightshade family include tomato potato chilli pepper and eggplant. Tomato (Lycopersicon esculentum Miller) often referred to as fruity vegetable" is second major vegetable crop produced in Pakistan (Mirza 2007). Tomato is considered as highly nutritious because of its high contents of vitamin A and C as well as lycopene natural antioxidant which is not found in the other solanaceous crops. It has niacin 0.712 mg calcium 31 mg and water 94.28 g per 100 g weight (Anonymous 2008). In Pakistan tomato is cultivated over an area of 58.196 thousand hectares with a production of 574.052 thousand tons annually (AGRISTAT 2013). Of the various constraints responsible for low yield tomato like other vegetables is also vulnerable to abiotic and biotic stresses (Abdel-Sayed 2006).

However fungal diseases particularly early blight caused by Alternaria solani is most common and destructive one causing great reduction in the quantity and quality of fruit yield wherever tomato is grown (Tewari and Vishunavat 2012). Early blight is usually characterized by the appearance of brown to dark brown necrotic spots having concentric rings on foliage stem and fruits. Concentric rings inside the spots produced target board effect (Singh 1987). Alternaria (Nees. ex. Fr.) is a dematiaceous fungus commonly isolated from plants soil food and indoor air environment. The production of melanin-like pigment on target host is one of its distinguishing characters (Bell and Wheeler 1986). Alternaria sp. can easily be recognized by the morphology of their large catenate conidia formed in acropetal chains or solitary and multicelled having long apical beak. In spite of this A.

Solani bears smooth dark olive or olive-brown muriform solitary conidia with a long filiform beak ended with a small pore (Ellis 1976). Such uniqueness of Alternaria spp. has made challenging and exciting ground for exploring its morphological and cultural variations.

Tomato early blight is favored by warm temperature and extended periods of leaf wetness from dew rain fall and crowded plantation. The plants are more susceptible to infection by the disease during fruiting period (Cerkauskas 2005; Momel and Pemezny 2006). An understanding of the role of environmental conditions and its consequence on infection and survival of the pathogen is needed to develop disease management practices. Earlier investigations reported the influence of environmental factors such as temperature illumination relative humidity (RH) and composition of the culture media on morphology of conidia produced in vitro of various fungi (Vieira 2004).

In recent years tunnel farming has gained popularity in Pakistan as it provides effectual way of protecting crop from low temperature both in spring and fall. Tomato like other vegetables cultivated in tunnels gives early crop as compared to field grown tomato. Crops grown in tunnels are as susceptible to pest and diseases as those grown under field conditions. Apart from other diseases early blight of tomato in tunnels as well as in field is the main restraining factor for tomato production. The losses from this disease may increase significantly if no protective measures are adopted well ahead of time. However the use of fungicides one of the most effective and conventional method may help in reducing disease spread if applied to greenhouse and tunnels in addition to field. Various fungicides including captafol mancozeb benomyl carbendazim copper oxychloride and Mancozeb have been used to control tomato early blight (Mate et al. 2005).

Recently an ecofriendly biocontrol agent Bacillus subtilis has received much attention by both conventional and organic farmers to suppress plant diseases (Zitter et al. 2005; Romero et al. 2007). Biological control of A. solani with Trichoderma sp. has been proved more effective and environment friendly (Mukerji and Garg 1988; Gardener and Fravel 2002). Trichoderma species besides inhibiting growth of fungi also promote growth and development of plant (Samuels 2006). The present study has therefore been undertaken with the objective to screen tomato varieties determine the influence of defined environmental factors on the morphology of A. solani conidia and to evaluate the efficacy of fungicides and biological agents against pathogenic isolate of early blight of tomato under laboratory and tunnel cultivation.

Materials and Methods

An experiment was carried out during 2011-2012 to observe the severity of early blight disease in natural conditions on six local tomato varieties viz. Sahal Reograndi Salar Roma Nagina and Packit grown under walk-in tunnels of vegetable research area Faculty of Agricultural Sciences and Technology Bahauddin Zakariya University Multan.

Isolation and Identification of Culture

The infected tomato leaves showing distinctive symptoms of leaf blight were collected from experimental area. An isolate of A. solani was obtained by tissue segment method (Rangaswami 1958) on potato dextrose agar (PDA) medium. The infected leaves were cut into 12 cm pieces surface sterilized with sodium hypochlorite solution (1%) for 2 min rinsed thrice with sterile distilled water blot dried and placed on PDA medium amended with streptomycin (250 mg/L). The petri plates were incubated at 272C for 5 days. Fungal mycelia growing out from segments were subsequently transferred to fresh PDA plates. Pure culture was obtained by re-culturing of isolated fungi through single spore technique (Choi et al. 1999) and maintained as stock culture on Agar slants at 4C for further studies. Pathogen was identified following the cultural and morphobiometric characteristics criteria (Ellis 1971; Barnet and Hunter 1972).

Cultural characteristics were observed directly by pigmentation on medium and mycelial growth pattern on PDA plates while sporulation was recorded by slides from 10-day-old culture under the microscope. Ocular and stage micrometer were used to measure the size of conidia.

Pathogenicity Test

Pathogenicity was carried out by atomizing the conidial suspension (5 A- 106 conidia/mL) at the rate of 8-10 mL/plant prepared from 10-day-old culture on to the 2- month old seedlings of moderately susceptible tomato variety Reograndi grown under disease conditions. Conidia were harvested by dislodging the surface of fungal colony with glass rod transferred to sterile distilled water and filtering through sterile cheese cloth. The resultant suspension was then adjusted 5 x 106 conidia/mL with the help of (Neubauer improved) haemocytometer. Control plants were sprayed with sterile distilled water. All test plants were covered with polyethylene bags for 48 h to retain humidity. The test plants were then uncovered and kept in green house. The experiment was run in quadruplicate with eight seedlings per replication. Observations were recorded after seven days for symptom development and re isolation were made from test plants thus fulfilling the Koch's postulates.

Effect of Culture Media

The cultural characteristics of the pathogen were studied on two solid media. Petri plates (9 cm) containing 20 mL of each media. Potato dextrose agar (Peeled potato = 200 g Dextrose and Agar Agar = 20 g each) and host leaf extract agar (Healthy tomato leaves = 200 g and Agar Agar = 20 g) were seeded with 0.4 cm diameter disc from 10 days old culture of A. solani isolate. Three replications with three plates per replication were maintained for each media and the inoculated plates were kept in completely randomized design at 272C for 7 days. Colony diameter was recorded after 24 h.

Effect of Temperature Light Regime and pH on Mycelial Growth of A. solani (In Vitro)

Petriplates comprising 20 mL of Potato dextrose Agar medium were seeded with mycelial discs of 0.4 cm in diameter from margins of 7 days old actively growing colony of A. solani. The inoculated plates were incubated at different temperature ranges of 20 25 30 35C under cool illumination. Similarly for light effect PDA plates were exposed to continuous light darkness and 12 h light (Philips daylight fluorescent lamp 20W TLT 75RS) altered with 12 h dark and maintained at 25C in controlled environment. While in case of determining best pH A. solani isolate was inoculated on PDA medium with pH adjusted to 5.5 6.5 and 7.5 with the help of digital pH meter by adding 0 .1N sodium hydroxide and 0.1N hydrochloric acid and buffered with standard phosphate buffer. All the inoculated plates were kept in incubator at 252oC for 7 days. The experiment was carried out in a completely randomized design with three replications. The colony diameter was measured daily for 7 days.

In vitro Evaluation of Fungicides and Biological Control agents on Growth of A. solani

Poisoned Food Technique (Dhingra and Sinclair 1985) was used for in vitro evaluation at different concentrations (1 2 and 3 g/L by weight of formulation) of the fungicides namely Bavistin (carbendazim) Ridomil Gold MZ (metalaxyl + mancozeb) and Topsin M (thiophenate methyl) against A. solani. After autoclaving the PDA media was amended with certain volumes of fungicides at 45oC before pouring to sterilized petri plates. The petri plates were aseptically inoculated in center with 0.4 cm mycelia plugs taken from growing margins of 10-day-old A. solani culture. Each treatment was replicated thrice and plates without any fungicide were used as check. All plates were incubated at 272oC for seven days. Two isolates of biological control agent T. harzianum and T. viridae isolated on Trichodermaspecial medium (Elad and Chet 1963) from rhizosphere of cucumber and chillies were used against A. solani on PDA by dual culture technique (Morton and Strouble 1955).

Discs of mycelia from fungal culture plate were placed on the edge of each PDA plate containing A. solani at opposite direction in triplicate with control without any isolate of Trichoderma and these plates were incubated at 252oC until the A. solani covered whole plate in control. Both fungicide and biological control agent's trial were subjected to Completely Randomized Design in triplicate. Efficacy of fungicides and Trichoderma isolates was determined by measuring linear growth (cm) on daily basis and data was expressed as percent inhibition over control using formula suggested by Sundar et al. (1995).

Equation

Screening of Tomato Varieties

Six tomato varieties (Sahal Reograndi Salar Roma Nagina and Packit) were sown in nursery on 15th October 2011 and were transplanted to plastic tunnel with a dimension of 6 ft height 12 ft width and 20 ft length covered with UV polyethylene film of 10 mm thickness on 15th November 2011. The plant to plant and bed to bed distance was of 30 cm and 70 cm respectively on both sides of bed which was covered with black plastic mulch. All recommended agronomic practices were carried out. The experiment was conducted in randomized complete block design with four replications while each replication contains six plants. Symptoms produced by the pathogen were observed on regular basis and experiment was repeated twice.

Disease Severity on Leaves and Fruits of Tomato

Infected leaves and fruits of tomato were observed for detection of early blight disease severity. Ten leaves and fruits were selected randomly from each replication and tagged. The data of disease severity was assessed in randomized complete block design with four replications by using modified 0-5 disease rating scale of Mayee and Datar (1986) for leaves where 0= No symptoms on leaves 1= 0- 5% infection on leaves 2= 6-20% infection on leaves 3= 21-40% infection on leaves 4= 41-70% infection on leaves 5= greater than 71% infection on leaves. Whereas 0-8 rating scale suggested by Dillard (1989) for fruits where0= No apparent disease 1= 1-12.5% 2= 12.5-25% 3= 25-37.5% 4= 37.5- 50% 5= 50-62.5% 6= 62.5-75% 7= 75-87.5% 8= 87.5- 100% were used and Percent Disease Index (PDI) was expressed by using formula suggested by Wheeler (1969).

Formula

In vivo Evaluation of Fungicides and Bio Control Agents

Laboratory tested fungicides and Trichoderma isolates were applied in tunnel on susceptible tomato variety Roma that showed maximum percentage of disease severity on leaves and fruit preceding year. The same nursery and transplanting procedure as described above was adopted. After four weeks of transplanting tomato plants were sprayed with (100 mL/plant) of A. solani suspension at the concentration of (5 A- 106 conidia/mL). After 10 days of inoculation these plants were sprayed (150 mL/plant) with the fungicides Topsin M Bavistin and Ridomil Gold MZ at the concentration of 2 and 3 g/L of water and bio control agents T. harzianum and T. viridae at the concentration of 107 and 108 conidia/mL adjusted with the aid of haemocytometer. While the control treatments were sprayed with A. solani suspension only. The same was repeated after the development of fruit. The experiment was run in a randomized complete block design with three replications.

Data of disease severity was taken after 15 days of fungicides and bio control agent's application to assess the effect of time on the antagonist activity (El-Katatny and Emam 2012).

Statistical Analysis

Data were analyzed by following ANOVA to differentiate statistically significant means. Fisher's Least Significant Difference (LSD) test was used to compare and separate means at 5% level of significance (Steel et al. 1997) by using statistical software Statistix 8.1.

Results

Microscopic study revealed that the conidiophores of A. solani isolate were straight or flexuous and brown to olivaceous brown. The conidia were solitary straight or slightly flexuous oblong or ellipsoidal tapering to a beak pale or olivaceous brown smooth 150-300 m in length 13-20 m thick in the broadest part with 8-10 transverse and none or few (1 to 4) longitudinal septa. The beaks were flexuous pale and sometimes branched.Two media namely Potato Dextrose Agar (PDA) and Host Leaf Extract (HLE) agar were used for the isolation and assessment of the best media for the growth of A. solani. Fungus grew on both media but growth was better on PDA (8.64 cm) compared to HLE agar with (6.52 cm).

The color of colony was observed dark brown on PDA and light brown on host leaf extract agar. The daily data of mycelial growth at different temperature was statistically significant. The growth of A. solani started slowly and then progresses day by day. But the significant growth (7.56 cm) was observed at 25C followed by 35C 20C and 30C with 7.28 5.36 and 5.00 cm growth respectively. Mycelial growth of the fungus was statistically significant at different light conditions. The preliminary studies carried out in the present investigation with A. solani indicated significant growth and sporulation when the inoculated plates were exposed to continuous light followed by continuous dark and conditions (12 h light alternated with 12 h dark) with 9.00 7.71 and 5.54 cm growth respectively. The optimum pH for the growth of A solani was in the range of 5.5 (HCl) 6.5 (PDA) and 7.5 (NaOH).

However less growth of the fungus was recorded at pH 5.5 and maximum on pH 6.5 with 5.89 cm and 8.34 cm growth respectively and pH 7.5 showed intermediate results with 6.79 cm growth and data of growth on three different pH concentrations was statistically significant.

This shows that A. solani prefers acidic pH to alkaline pH indicating its acid tolerance (Table 1).

The percent inhibition of the growth of A. solani was significantly different at all fungicide concentrations compared to control. Bavistin was less effective at 1g/ liter with 17.65% inhibition while Ridomil Gold MZ was more effective at same concentration with 29.49 and Topsin M was intermediate with 29.41% inhibition. At concentration 2 g/L Ridomil Gold MZ was most effective with 47.06% inhibition followed by Topsin M and Bavistin with 41.18 and 23.53% inhibition respectively. Maximum growth of A. solani was inhibited at concentration i.e. 3 g/L by Topsin M with 64.71% inhibition followed by Ridomil Gold MZ and Bavistin with 52.94 and 35.29% inhibition respectively compared to control with 0% inhibition (Table 4). The growth of A. solani was checked in the presence of two antagonistic isolates of Trichoderma sp. The plates containing biological control agent reduced the tested fungus growth considerably.

Inhibition percentage was maximum (67.78%) with T. harzianum followed by 59.63% inhibition in T. viridae compared to control with zero percent inhibition (Table 2). At initial stage the data of disease severity on tomato leaves depicted that Nagina was resistant with only 3.75% severity followed by Sahal (5.38%) Packit (6.25%) Reograndi (18.75%) Salar (24%) and Roma (30.25%) with the passage of time disease spread continuously. At 20th day salar ranked first in susceptibility with 57% severity followed by roma reograndi sahal packit and nagina with 56.88 55.75 29.13 29 and 25.38% respectively. At 35th day it was observed that no line/variety were resistant to early blight disease. But maximum disease was on roma with 88% severity while less disease 66.13% was on nagina (Table 3).

Similarly disease severity on tomato fruit was also statistically significant as recorded by modified 0-8 disease rating scale suggested by Dillard (1989). During 5th day reograndi salar were resistant with 0% severity on fruits followed by low disease percentage 0.25 0.50 0.75 and 1.50% in roma packit nagina and sahal respectively. At 20th day the situation of severity was changed from previous data. Fruit of roma was damaged more with 19.50% followed by sahal salar packit reograndi and nagina with 18.38 16.75 11.75 9.50 and 7% severity respectively. At 35th day roma which showed highest severity on leaves also susceptible to pathogen attack on fruit with 70.50% severity while fruit of nagina was less affected (27.38%) by the pathogen (Table 3).

Fungicides concentrations which proved best in laboratory trial were applied in tunnel on susceptible tomato variety Roma that showed highest percentage of leaf and fruit affected. Three fungicides with two best concentrations (2 and 3 g/L) showed statistically significant results of disease inhibition on leaves and fruits compared to control. Bavistin with 65% severity at concentration 2 g/L was weaker compared to other two fungicides with 55.50% and

Table 1: Effect of temperature light regime and pH on in vitro growth of A. solani

Days###Temperature Ranges (oC)###Light Conditions###pH Variation

###20###25###30###35###Darkness Continuous light###12 h light 12 h dark 5.5 (HCl) 6.5 (PDA)###7.5 (NaOH)

1###0.24 x 0.64 v###0.72 u###0.60 w###0.80 t###1.03 s###0.63 u###0.70 r###1.20 p###0.90 q

2###1.20 s###1.20 s###1.20 s###0.96 t###1.83 p###2.40 o###1.13 r###1.67 o###3.10 l###2.71 m

3###2.16 r###3.20 n###3.32 m###1.20 s###2.46 n###3.60 l###1.66 q###2.34 n###4.17 i###3.57 k

4###3.00 p###3.60 l###3.96 j###2.64 q###3.71 k###5.71 f###2.80 m###3.10 l###5.30 f###4.16 i

5###3.16 o 4.68 f###4.36 h###4.20 i###5.38 h###6.46 e###4.06 j###3.94 j###6.38 d###5.06 g

6###3.84 k###5.60 c###4.60 g###5.36 d###6.49 d###8.11 b###4.46 i###4.76 h###7.40 b###5.98 e

7###5.36 d###7.56 a###5.00 e###7.28 b###7.71 c###9.00 a###5.54 g###5.89 e###8.34 a###6.79 c

Table 2: Efficacy of different concentrations of three fungicides against A. solani (in vitro)

Doses###Inhibition Percentage

###Topsin M###Ridomil Gold MZ###Bavistin###T. harzianum###T. viridae

1 g/L###29.41 g###29.49 f###17.65 i###-###-

2 g/L###41.18 d###47.06 c###23.53 h###-###-

3 g/L###64.71 a###52.94 b###35.29 e###-###-

-###67.78ns###59.63ns

Table 3: Severity of early blight disease on tomato leaves and fruits in tunnel

Varieties###Disease severity on Leaves (%)###Disease severity on Fruits (%)

###Days###Days

###5###10###15###20###25###30###35###5###10###15###20###25###30###35

Reograndi 18.75 c 27.00 c 40.50 b 55.75 a 70.50 a 77.75 a 82.38 b 0.00 b 1.75 cd 3.75 d 9.50 e 17.13 e 35.63 c###53.38 d

Salar###24.00 b 31.00 b 38.63 c 57.00 a 66.88 c 74.13 b 84.13 b 0.00 b 2.63 b###6.00 c 16.75 c 27.25 c 43.63 b###67.50 b

Roma###30.25 a 39.75 a 48.38 a 56.88 a 68.38 b 77.38 a 88.00 a 0.25 b 4.25 a###9.00 b 19.50 a 39.88 a 60.25 a###70.50 a

Nagina###3.75 e 11.63 f 15.88 f 25.38 c 37.13 f 52.00 e 66.13 e 0.75 ab 1.50 d###3.38 d 7.00 f###12.50 f 20.00 e###27.38 f

Packit###6.25 d 13.75 d 19.88 d 29.00 b 40.88 e 55.13 d 69.00 d 0.50 b 2.25 bc 5.63 c 11.75 d 17.63 d 24.13 d###34.00 e

Sahal###5.38 d 12.75 e 17.88 e 29.13 b 44.38 d 66.00 c 76.88 c 1.50 a 4.00 a###9.75 a 18.38 b 30.88 b 43.50 b###57.50 c

LSD0.05###1.206###0.878###1.032###1.360###1.357###0.889###2.707###0.800 0.653###0.666###1.073###0.361###0.885###0.657

49.50% severity on leaves in Ridomil Gold MZ and Topsin M respectively. Topsin M showed exceptional control at 3 g/L concentration with only 24% disease severity followed by 30.50% and 34% severity in Ridomil Gold MZ and Bavistin respectively compared to control 85.50% severity. Similar to control of disease on leaves all fungicides decreased the severity at both concentrations. The best control was exhibited at 3 g/L concentration. Minimum severity (27%) was observed on plants sprayed with Topsin M followed by Ridomil Gold MZ and Bavistin with 39 and 45% severity respectively compared to control 91% (Table 4).

Keeping in view the inhibition of A. solani with two Trichoderma isolates in laboratory both T. harzianum and T. viridae were applied on leaves and fruits of tomato in tunnel with two different concentrations 107 and 108 conidia/mL. Growth of the fungus on leaves and fruit after spraying both isolates was statistically significant. T. harzianum decreased pathogen growth at both the concentrations compared to T. viridae. Tomato plants showed 65 76% and 41 49.50% severity on leaves sprayed with 107 and 108 conidia/mL concentrations of T. harzianum and T. viridae respectively compared to control with 86.50% severity. Similarly disease severity on fruit was 59.50 80% and 53.50 66% at 107 and 108 conidia/mL concentrations of T. harzianum and T. viridae respectively compared to control with 93.50% severity (Table 5).

Discussion

A. solani was initially identified on the basis of symptoms. Presently symptomatology is not a reliable method for detection of fungal disease but it is an initial step in disease diagnosis (Batool et al. 2011) while modern detection techniques e.g. PCR were found more reliable for disease identification. Pathogenicity was carried out by applying conidial suspension on seedlings for confirmation of pathogen association with host. Similar approach has been followed by different scientists. Vloutoglou and Kalogerakis (2000) and Castro et al. (1999) performed the pathogenicity test with conidial suspensions. Conidial suspensions of A. solani were more effective for preparation of inoculum load and pathogenicity test against tomato and potato (Rotem

Table 4: Effect of different concentrations of fungicides on early blight severity (%) on leaves and fruits of Roma in tunnel

Doses###Fungicides

###Bavistin###Ridomil Gold###Topsin M###Mean###Bavistin###Ridomil Gold###Topsin M###Mean

###Leaf###Fruit

Control###85.50 a###85.50 a###85.50 a###85.50 A###91.00 a###91.00 a###91.00 a###91.00 A

2g / Liter###65.00 b###55.50 c###49.50 d###56.67 B###71.00 b###61.00 c###54.00 d###62.00 B

3g / Liter###34.00 e###30.50 f###24.00 g###29.50 C###45.00 e###39.00 f###27.00 g###37.00 C

Mean###61.50 A###57.17 B###53.00 C###69 A###63. 67 B###57.33 C

Table 5: Effect of different concentrations of biological control agent's on early blight severity (%) on leaves and fruits of

Roma in tunnel

Doses###Biocontrol Agents

###T. harzianum###T. viridae###Mean###T. harzianum###T. viridae###Mean

###Leaf###Fruit

Control###86.50 a###86.50 a###86.50 A###93.50 a###93.50 a###93.50 A

107###65.00 c###76.00 b###70.50 B###59.50 d###80.00 b###69.75 B

108###41.00 e###49.50 d###45.25 C###53.50 e###66.00 c###59.75 C

Mean###64.17 B###70.67 A###68.83 B###79.83 A

1994). Light spectrum temperature pH and nutrition are major factors that influence the sporulation of A. solani. The maximum growth was observed at 250C continuous light condition and 6.5 pH. While minimum growth at 300C 12 h light and 12 h dark condition and 5.5 pH. Sodlauskiene et al. (2003) and Gemawat and Ghosh (1979) tested the isolate of A. solani on particular temperature and pH respectively.

Out of two media used both resulted in good sporulation of the pathogen but PDA performed better compared to host leaf extract agar. Similarly Vieira (2004) used PDA and plants extracts media to induce sporulation of Alternaria spp. Three fungicides at three different concentrations and two species of biocontrol agent Trichoderma were tested on PDA under laboratory condition to check the growth of A. solani. Ridomil Gold MZ was efficient at concentration 1 g and 2 g/L and resulted in decreased sporulation of the pathogen. But at higher concentration i.e. 3 g of fungicide/L Topsin M depicted excellent inhibition of the pathogen compared to others. But the results showed that dose 3 g/L of water of all the fungicides inhibited growth of the pathogen significantly. Different fungicides namely Zineb iprodione copper oxychloride dithianon mancozeb carbendazim captafol and thiophanate-methyl were tested against

A. solani and Mancozeb and reduced effective (77%) growth inhibited followed by captafol while Dithane M-45 was most effective to control the fungus (Elad et al. 1995). Effectiveness of mancozeb (0.2%) against early blight fungus of tomato was confirmed by Choulwar et al. (1989) Singh et al. (2001) while Babu et al. (2001) also found Mancozeb (0.2%) very effective against A. solani followed by Captafol (0.2%). Trichoderma species due to its antagonistic activity are considered as potential biological control agents against numerous plant pathogenic fungi (Mohamed and Haggag 2006). The most studied fungal biocontrol agent (Trichoderma spp.) has been marketed as biopesticides biofertilizers and soil amendments commercially for many years (Harman et al. 2004; Lorito et al. 2004). T. harzianum and T. viridae were used. T. harzianum showed maximum inhibition percentage compared to T. viridae.

Mukerji and Garg (1988) described that Trichoderma are more reliable in biological control because it is easy to isolate and culture while its enzyme system along with production of different antibiotics increase its efficiency to control different pathogens. Raziq and Ishtiaq (2010) confirmed that different fungicides and Trichoderma species effectively reduced the growth of A. solani under laboratory condition. Many workers confirmed that Trichoderma sp. control the pathogen growth due to the production of extracellular enzymes antifungal metabolites and antibiotics (El-Katatny et al. 2006; Montealegre et al. 2010). El-Katatny et al. (2006) confirmed the antagonistic activity of two isolates of T. harzianum (T3 and T24) against phytopathogens. Both T. harzianum strains were generally effective at low concentration of 106-108 conidia per ml. These concentrations are even lower than the recommended concentrations of other biocontrol agents (Wang et al. 2008) thus considered suitable for commercial use.

Therefore the use of Trichoderma isolates offers a potential safe and efficient mean alternative to fungicides in treatment of different fungal diseases of tomato fruits. However such measures should be adopted in future to provide these isolates for the control of pathogens in field condition instead of fungicides to minimize the human health hazards and environmental pollution.

Among selected varieties/lines Reograndi and Salar showed resistance during early stage of infection but high disease severity was observed at later stage. Maximum infection of fungus was observed on the leaves and fruit of Roma and Packit respectively while Nagina showed resistance against leaves and fruit infection. The yield (quality and quantity) of Solanaceous crop can be enhanced by cultivation of resistance varieties against nematode bacteria viruses and fungus which are more effective and environment friendly (Abbas and Hameed 2012). Serological and molecular techniques are also reliable for the screening of resistance source.

Conclusion

Resistant or tolerant local tomato varieties biological and chemical control strategies against A. solani may play a vital role in reducing yield losses and thus may increase the income of farmer. It is therefore suggested that farmers along with resistant lines/varieties should employ recommended fungicides and bio-control agents to make tomato less vulnerable to early blight pathogen both under conventional and tunnel farming.

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