Disease Free Seed Production of Radish

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Disease Free Seed Production of Radish


Radish (Raphanus sativus L.) is an important vegetable of both tropical and temperate regions. It’s fleshy edible root is rich in Ca, K, P, and vitamin C (Srinivas and Naik, 1990). It is the third major vegetable crop next to potato and brinjal in Bangladesh in term of both area and production. In Bangladesh radish is cultivated in an area 171 ha producing 202 thousands metric tons of fleshy edible roots (BBS. 2008).

Depending upon flowering radishes may be classified as annual (tropical) and biennial (temperate). In Bangladesh condition the tropical annual type varieties are suitable for seed production; the temperate biennial varieties do not produce seed (Rashid and Singh 2000). The average seed yield ranges from 500 to 1000 kg/ha; higher yields up to 1400 kg/ha can be obtained under favorable condition (Rashid and Singh, 2000).

Unavailability of quality seed is the main constraint for potential radish production. To exploit the benefits of hybrid radish Bangladesh has to depend on imported seeds with a cost of our hardly earned foreign currency. Again the farmers for purchasing seeds spend a large share of cultivation cost. Later in the field they are fighting with diseases may be soil or seed borne. Among the diseases, Alternaria blight caused Alternaria brassicae and A. brassicola are the most devastating ones which cause serious yield losses major constraint for high quality radish seed production in Bangladesh. This disease cause 30-40% yield loss of the crop annually in Bangladesh (Anonymous 1997). Alternaria brassicae and A. brassicola appears on pods as small, blackish, circular lesions which eventually cause the death of the pod (Prasada et al.1970). Severe foliage infection causes reduction of flower which results in production of diseased pods and seeds (Chand and Jatian 1969). The disease is enhanced by warm, wet weather with an optimum temperature between 25 and 30°C. Plants wound inoculated with A. brassicicola develop symptoms most quickly at 250 C, while seedlings from infected seeds develop symptoms most quickly at 30C (Bassey and Gabrielson 1983). Infected seeds, with spores on the seed coat or mycelium under the seed coat, are likely the main source of transport for these pathogens. Spores are disseminated by wind, tools and animals. The fungus can survive in susceptible weeds or perennial crops (Chupp and Sherf 1960; Rangel 1945; Humpherson-Jones 1989).

Control of Alternaria blight in crucifers is very difficult due to various sources of inoculums and wide ranges in conidial dispersal and uncertainty in the level of disease, several fungicides could effectively control the disease. Iprodione has been proved very effective in controlling the Alternaria blight of mustard in Bangladesh (Meah et al. 1992). In controlling Alternaria blight of radish Rovral 50 WP (Iprodione) @ 0.20% found as an effective chemical by several investigators (Islam et al. 2007, Shrestha 2005, Khoda et al. 2003, Ayub 2001).

In recent years, an increasing consciousness about environmental pollution due to pesticides and development of fungicide-resistant strains in plant pathogens has challenged plant pathologists to search for eco-friendly tools for disease management. Fungi in the genus Trichoderma have been known since at least the 1920s for their ability to act as biocontrol agents against plant pathogens (Harman, 2006). These fungi frequently increase plant growth and productivity (Lindsey and Baker, 1967) either in the presence (Chang et al. 1986) or absence (Lindsey and Baker, 1967) of other microorganisms and that they can induce disease suppression in soils (Chet and Baker 1981). In case of controlling Alternaria blight, treatment with biocontrol agents like Trichoderma initiate in the plants a number of biochemical changes which can lead to plant defense response (Sharma et al. 2010). Again Trichoderma application has positive response to the germination, growth and yield of radish (Phuwiwat and Soytong 1999, Windham et al. 1986, Baker 1986).

Apart from conventional fungicides and microbial biocontrol agents, plant products/extracts have been found effective against wide range of pathogens (Amadioha, 2003; Bowers and Locke, 2004; Suratazzaman et al. 1994). It may be a possible source of non-phytotoxic, systemic and easily biodegradable alternative pesticides (Singh 1994; Qasem and Abu-Blan 1996; Mason and Mathew 1996). Several studies reported direct effect of neem leaf and fruit extracts on target pests and pathogens (Eppler, 1995; Amadioha, 2000). Induced resistance of crop was reported by the use of neem plant extract (Paul and Sharma, 2002; Guleria and Kumar, 2006). No effort has been made to use the plant extracts and bio-agents as eco-friendly components for effective integrated management of Alternaria blight of radish for disease free seed production. However, information on the control of Alternaria blight, particularly on radish seed crop, is limited in this country. Therefore, the current study was designed to achieve the following objectives:

· to find out the effect of fungicide, plant extract and Trichoderma individually and in combination for integrated management of Alternaria blight for production of healthy and disease free seeds of radish.


Control of Alternaria blight by applying fungicides

Lot of investigations has been done in controlling Alternaria blight of crucifers. Several related review of literatures are cited below:

Maude (1977) evaluated that the surface applications of contact fungicides did not eliminates seed infection, where soaking in Thiram reduced infection by 99% but adversely affected germination, while application of the Rovral to seeds at 10g/kg eradicated infection without affecting germination. Additionally, spray trials indicated that Rovral reduced seedpod infection and prevented the development of seed- borne infection.

The effectiveness of different fungicides for the control of white rust and Alternaria leaf spot of mustard was evaluated by Gupta et al. (1978). They recorded disease intensity on five randomly selected plants per plot estimating the severity of infection on leaves borne on lower 213 portion of the plants. They recommended Brestan (Carbendazim) (0.1%) for its best result for the control of Alternaria leaf spot followed by Miltox(0.3%). They used only two sprays fortnightly starting at the age of two months of the crop.

Nene and Thapliyal (1979) have recommended three or four sprays with any one of the dithiocarbamate fungicides such as Mancozeb (Dithane M – 45) or Zineb (Dithane Z -78) and Iprodione for the control of Alternaria leaf blight of crucifers caused by A. brassicae.

Humpherson and Ainsworth ( 1983) suggested that three sprays of Rovral at 0.5-10.0 kg a.i. per ha applied on Brassica oleracea seed crops at three-week intervals at young green siliqua stage to control pod infection caused by A. brassicicola. Their findings demonstrated that seed yield was increased and the spray improved the germination. They also reported that Bordeaux mixture was also as effective as Rovral when disease levels were low but ineffective when infection pressure was severe.

Sharma (1984) conducted an experiment in the field on the efficacy of 10 fungicides. The best results in controlling Alternaria sp, infection of mustard were obtained with 4 sprays of Dithane M-45 (Mancozeb) at l0 days intervals starting from onset of disease. Daconil (0.1%), Difolatan Q3%) and Dithane Z-78 (0.2%) were found effective. The fungicides reduced the infection rate by 16.6-30.1%.

Ryan et al. 1984 reported that Alternaria brassicae and A. brassicicola were prevalent in Brassica crops from August onwards which was successfully controlled by Fosetyl aluminium, Metalaxyl + Mancozeb, Cyprofuram and Proamocarb. Meah et al. (1985) studied the effects of 5 foliar fungicides viz., Dithane M-45, Duter, Benlare, Tri-Miltox, Forte and Cupravit at single dose against Alternaria brassicae on mustard in two cropping seasons under natural conditions. All the fungicides sprayed at 8 days intervals starting from 30 days of sprouting and continued up to crop maturity, significantly reduced defoliation, leaf spot severity and number of spot on siliqua and increased the yield.

For the management of Alternaria blight of mustard, different fungicides were evaluated by Shivpuri et al. (1988). Six fungicides namely Rovral (Iprodione), captafol, Dithane M-45(Mancozeb), Thiram, Blitox-50 (Copper oxychloride), Bavistin (Carbendazim) were applied to mustard infected by Alternaria brassicae in field trials in Rajasthan, India. They reported that all of the fungicides controlled the disease but copper oxychloride was found phytotoxic. The best treatment was Rovral followed by captafol and Dithane M-45.

Control of A. brassicae in radish, cauliflower, cabbage and turnip was positively correlated with persistence of fungicides. Captafol (0.2%) provided maximum control and persistent the longest on all the hosts. Mancozeb, Carbendazim and Copper oxychloride at 0.3% exhibited highly differential behavior against the disease due to varying levels of persistence, Copper oxychloride enhanced disease intensity on all the hosts after a brief period of protection (Thind and Jhooty 1988).

The most effective control of Alternaria brassicae as obtained with Iprodione ( as Rovral) followed by Mancozeb ( as Dithane M -45), Zineb( as Dithane Z -78),Captan, Benomyl (as Benlate), Thiophanate-methy (as Topsin M) and Carbendazim ( as Bavistin) at l0 day’s interval (Mondale et al. 1989).

Alternaria brassicae causes severe damage to cauliflower, producing different symptoms including damping off, seedling blight, stump rot, leaf spot, leaf blight and curd rot. Three sprays with 0.2% captafol were found effective to control leaf spot disease of cauliflower caused by Alternaria brasssicae under field condition. Seven fungicides namely, Emisan-6, Bavistin, captafol, Cuman-L, Difolatan, Dithane M-45 and Dithane Z-75 were tested under field conditions for suggesting economically viable control measures acceptable to farmers. Their findings concluded that three sprays with Difolatan (0.2%) distinctly scored higher over other fungicides’ both in terms of additional crop yield and net profit with a benefit cost ratio of 13:2 (Singh et al. 1990a).

Meah et al. (1992) observed the effects of frequencies, doses and time of application and their combination in controlling Alternaria blight of mustard in two consecutive cropping seasons under natural conditions. They found Rovral (1.0 Lit/ha) significantly reduced disease severity and increased seed yield by 147% over control when applied two times commencing from fruiting stage (50 days age) at l0 days intervals onward.

Babadoost (1993) evaluated three systemic fungicides for their potentiality to control Alternaria disease of Brasssica seed crops. It appeared that two applications of either Iprodione (1.2 g a.i. /liter), Anilazine( 3.6 g a.i. / liter) or Chlorothalonil (1 .4g a.i. /liter) significantly reduced the severity of disease of brussels sprouts seed plants caused by A. brassicae and A. bassicicola. Iprodione and Anilazine also significantly reduced the incidence of Alternaria sp. on surface sterilized seeds. Percentage of seed germination was negatively correlated with the incidence of Alternaria sp.

Seven fungicides were evaluated by Ayub et al. (1996) to control Alternaria blight of mustard caused by A. brassicae and A. brasiicicola. Fungicides namely carbendazim (as Bavistin) and Benomyl (as Benlate) at 0.1%, Zirum (as Cuman), Mancozeb (as Dithane M -45), Fenlin hydroxide (as Duter), Iprodione (as Rovral) and copper salts + Mancozeb (as Trimillox forte) at 0.2% were applied three times as foliar spray to plant at 40, 50 and 60 days old plants. The findings of the experiments demonstrated that Rovral was the best fungicide in reducing disease severity, increasing 1000 seed weight and seed yield. Maximum reduction of disease severity and increased yield was achieved when spray was applied on 40 days old plants.

Minimum Alternaria blight infection with Rovral (Iprodione) followed by Difolaran (captafol), Indofil M-45 (Mancozeb + thiophanate-methyl) and Ridomil Mz (Mancozeb + Metalaxyl) in a field trial was recorded by Kumar (1996). Maximum yield was recorded with iprodione. Esiyok et al. (1996) studied Ridomil and Antracol on cauliflower to control Alternaria leaf blight. Their results suggested that good seed production obtained from three alternate applications of Ridomil (Metalaxyl) and Antracol (Propineb).

The lowest incidence of Alternaria blight was found in pots where Rovral (0.2%) was applied at pod and seed formation stages. It was also observed that the higher seed yield was achieved from the treatment mentioned above. Thousand seed weight was also higher in Rovral treated plots (Anon. 1997).

Efficiency of five fungicides on the development of Alternaria brassicae on the crucifers was investigated by Selmaoui et al. (1999) and observed that all the fungicides reduce significantly the degree of infection of the disease in comparison to control. However, Iprodione followed by chlorotalonil and mancozeb permit the most reduction of the injured leaves of the cabbage “Brassicae oleracea” than the thioaphanate-methyl and the benomyl.

Alternaria blight (Alternaria brassicae) disease in rapeseed-mustard was evaluated by Chaudhary (1999) to determine the effectiveness of fungicide Iprodion 50WP (Rovral) over a currently recommended fungicide Mancozeb 75 (Dithane M-45) to control Alternaria blight disease. Spraying fungicides Iprodion and Mancozeb on mustard variety Bikash 30, 45, 60 days after seed sowing reduced Alternaria blight severity by 27-38% on leaves and defoliation by 24-50%. Iprodion also increased intact green leaves by 210-419%, maturity days by 7-8%, 1000-seed weight by 16-26% and seed yield by 21-70% more than Mancozeb in mustard-toria. Spraying Iprodion at 45 and 60 days after seeding and 30, 45, and 60 days after seed sowing increased seed yield of toria by 126 and 153%, respectively over unsprayed treatment.

Four sprays of Iprodione (0.2%) gave the maximum control of Alternaria blight. Maximum yield was obtained in the treatment with Ridomil 0.5%, while it was lowest in the control (Pandya 2000). Ayub (2001) concluded that application of Rovral at 0.2% and Knowin at 0.1% were effective to control Alternaria blight (caused by A. brassicae, A, brassicicola) of cauliflower.

A field experiment was conducted from 1994/95 to 1996/97 in Rajasthan, India to evaluate the efficacy of different fungicides, viz., Mancozeb, Ridomil MZ, (Mancozeb + Metalaxyl), Captan, Rovral (Iprodione), Bayleton (Triadimefon) and Copper oxychloride, against Alternaria blight (Alternaria brassicae) and white rust (Albugo candida) of Indian mustard by Godika (2001). All fungicides significantly controlled both diseases, but Rovral was the most effective in controlling of Alternaria blight.

Khoda (2003) conducted an experiment to evaluate the effectiveness of five fungicides viz., Rovral 50 WP (0.20%), DithaneM -45 (0.25%), Ridomil (0.10%), Bavistin (0.25%) and Knowin (0.25%) to control stalk rot and Alternaria blight (Alternaria brassicicola) of cauliflower seed crop. The maximum reduction in the severity of stalk rot and Alternaria blight and highest increase in seed yield over control were achieved with Rovral 50 WP (0.20%), which was followed by Dithane M-45 (0.25%).

Mukherjee et al. (2003) observed that the percentage incidence of Alternaria blight was less on using both the fungicides iprodione and mancozeb as compared to control. Iprodione was observed to be more effective than the conventional fungicide mancozeb in three years of bioefficacy trial as mean incidence Alternaria blight was less in both leaves as well as in pods. Increase in yield of mustard seed in iprodione treated plots ranged from 24-59 percent as compared to that realized from control plots.

Fungicides Mancozeb and Iprodione reduced the disease significantly and increased the seed yield by 48% and 130%, respectively. The correlations between the disease severity and yield and yield components were negatively significant. Yield losses were estimated to be 32-57%. Seed infection was also significantly higher in water sprayed plots than in fungicide sprayed plots. There was negative effect of fungus infection on oil content and the losses in oil content were 4.2-4.5% (Shrestha 2005).

Islam (2006) evaluated effectiveness of six fungicides namely Rovral 50 WP (Iprodione) @ 0.20%, Dithane M-45 (Mancozeb) @ 0.45%, Ridomil MZ 68 WP (Metalaxil) @ 0.20%, Bavistin 70 WP (Carbendazim) @ 0.10%, Tilt (Propiconazol) @ 0.20% and Cupravit (Copper oxichloride) @ 0.70% as foliar spray four times at seven days interval starting from flowering stage against Alternaria blight of radish seed crop. Maximum reduction of the severity of Alternaria blight over control as well as yield and yield contributing character were achieved with Rovral 50WP as foliar spray in radish.

Control of Alternaria blight by applying botanicals

Meah and Hossain (1989) used botanical extracts to control leaf blight of mustard. They systematically recorded the reduced number of diseased leaves per plant, percent leaf area diseased, pods per plant and number of spots per pods in plots of mustard versa sonali, sambal and sampad that received spray of mustard leaf extracts of sambal leaves brought 34.0 and 68.32% reduction of leaf area diseased and percent pod infection, respectively over control.

Khan (1999) studied the effect of plant extracts (Allamanda, Bel and Neem) for the management of phomopsis blight or fruit rot of eggplant in field condition by spraying and observed that among the 3 plant extracts Alamanda was most effective than Bel and Neem extracts.

Kohinoor et al. (2003) studied the effectiveness of two fungicides viz. Rovral 50WP (.20% and .30%), Dithane M-45 (0.25%) and two botanicals namely Neem (Azadiracta indica) and Bishkatali (Plygonum hydropiper) to control stalk rot and Alternaria blight (A.brassicicola) of cauliflower seed crop. He concluded that Rovral @ 0.2% gave the best results in controlling Alternaria blight and to increase the seed yield of cauliflower. It was followed by Dithane M-45. Neem leaf was somewhat effective but it was not as effective as the fungicides.

Aqueous extracts of neem, mahogany, koromcha and garlic clove was observed by Kabir (2006) as foliar spray, singly or in combination to evaluate the efficacy and profitability compared to no treatment at all and foliar spray with Rovral 50WP @ 0.2% to control the Alternaria leaf blight disease in Broccoli cv. Green. All the treatments controlled the disease significantly giving higher yield compared to no treatment control. Though the 0.20% Rovral gave the best result both in terms of disease control and yield of broccoli, the combined application of garlic , neem and koromcha extracts performed as good as Rovral. Rather this organic cocktail gave a higher BCR (4.74) due to reduced production cost indicating that judicious combination of organic management practice promises a healthy broccoli production which is environment friendly, healthy and profitable.

Aqueous leaf extract of neem (Azadirachta indica Juss.) was observed by Guleria and Kumar (2006) to control of Alternaria leaf spot pathogen (Alternaria sesami) of sesame (Sesamum indicum L: Syn. S. orientale L). Treatment with this extract led to the changes in plant metabolism as leaves of the treated plants exhibited significantly high level of enzymes phenylalanine ammonia-lyase (PAL), peroxidase (PO) and content of phenolic compounds. Furthermore, germination of A. sesami spores was not significantly inhibited by neem extract. It is therefore, suggested that, protection of sesame plants against A. sesami by neem extract might be due to stimulation of plants natural defense response.

Singh and Singh (2007) evaluated aqueous leaf extracts (2.0%) of fifteen locally available plants in vitro against Alternaria lini, the causal organism of leaf and bud blight in linseed. Maximum inhibition was recorded with Azadirachta indica (67.7%) followed by Lawsonia inermis (63.0%), Datura metel (39.2%), Calotropis procera, Lantana camara (36.6%) and Citrus sp. (28.1%). The inhibitory effect was higher (97.7 to 50.2%) at higher (5.0%) concentration. Foliar spray of six plant extracts controlled the disease significantly and enhanced grain yield (48.5 to 9.4%), in the same order under field conditions.

Prasad (2008) found that lower doses of the leaf extract of neem (Azadirachta indica A. Juss.) and neem-based biopesticide cause an increase in root weight, plant height and number branches/plant of turnip (Brassica rapa L.), but their higher doses have inhibitory effect in M1 generation. A further improvement in the above traits is seen in M2. Crude neem oil treatment demonstrated as retarding effect on the above traits. All the three kinds of treatment were observed to delay in the time of flowering. The 40% concentration of the leaf extract and bio-pesticide is the most beneficial in this regard.

An experiment was conducted to evaluate six botanicals viz. Garlic extract, onion bulb extract, neem leaf extract, allamanda leaf extract, marigold leaf extract, bishkatali leaf extract for their efficacy against Alternaria Blight of Cauliflower seed crop (Islam et al. 2009). Fungicide Rovral and untreated control were also used in the trial. Highest disease reduction was recorded from neem leaf extract. The lowest PDI value (22.80) was recorded from Neem leaf extract but it was statistically similar to Rovral and Allamanda extract. Maximum seed yield was obtained from Neem leaf extract treated plot followed by Rovral, Garlic extract and Marigold leaf extract treated plots, respectively.

Biswas et al. 2009 tested in vivo and in vitro in controlling Alternaria blight of mustard with plant-extracts of Lantana camera and Eucalyptus hybrida, bulb of garlic (Alium sativum) and onion (Allium cepa), rhizome of ginger (Zingiber officinale) @ 10%; neem seed oil(Azadirachta indica) @ 0.05% and other eco-friendly chemicals i.e. salicylic acid (SAR compound) @ 2 ppm; and mancozeb @ 0.2%,. Fungicidal treatment with Mancozeb 75% WP @ 0.20% was proved its superiority among all the treatments and a range of 22 to 72% reduction in disease severity and 6 to 44% increases in yield were observed with different plant extracts.

Control of Alternaria blight by applying Trichoderma

A total of 13 isolates of Trichoderma virens were isolated, tested and observed by Intana et al. (2005) to control mycelia growth of Alternaria brassicicola that causes Chinese kale leaf spot where isolate T-ST-01 and T-NST-01 found best. They also observed that use of antifungal metabolite is effective both in laboratory and glasshouse conditions. Disease severity of Chinese kale treated with antifungal from extracted T-ST- 01 were 22.5 and 32.0% when measured in laboratory and glasshouse conditions, respectively, while the severity in the control treatment (2% methanol) was found to be 89.5 and 84.5%, respectively.

The pentyl pyrone, purified from an ultraviolet-induced mutant strain of Trichoderma harzianum T-35-co4 at the concentrations of 50 and 100 mg L-1 were tested by Intana et al. 2008 for the inhibition of mycelial growth and spore germination of Alternaria brassicicola, a causal agent of leaf spot on Chinese-kale. They observed complete (100%) inhibition of mycelial growth of A. brassicicola at 100 mg L-1 of pentyl pyrone. They also observed that when Chinese-kale leaf was sprayed with 100 mg L-1 of pentyl pyrone, the disease severity of leaf spot was found in laboratory and under glasshouse conditions were 6.5 and 19.0% respectively, while those of a control treatments (2% methanol) were 88.0 and 92.5%, respectively.

Karthikeya et al. (2008) studied three antagonists: Pseudomonas fluorescens (Pf1), Bacillus subtilis and Trichoderma viride, alone and in combination for suppression of onion leaf blight (Alternaria palandui) disease under glasshouse and field conditions and the average mean of disease reduction was observed 24.81% for single strains and 42.44% for mixtures. In addition to disease suppression, treatment with a mixture of antagonists promoted plant growth in terms of increased plant height and ultimately bulb yield. They also observed that though seed treatment of either single strain or strain mixtures alone could reduce the disease, subsequent application to root, leaves or soil further reduced the disease and enhanced the plant growth. In reducing the disease and in promoting plant growth and bulb yield both in greenhouse and field tests, Pseudomonas fluorescens Pf1 + Bacillus subtilis + Trichoderma viride was found most effective in comparison to the other treatments.

Control of Alternaria blight by integrated management

Chattopadhay (1999) observed that integration of carbendazim spray with KCl soil application at the recommended dose of N fertilizer was the most effective treatment in disease reduction with corresponding increase in yield. The treatment A. sativum bulb extract spray + KCl soil application at recommended dose of N ranked second in disease reduction and yield performance.

The efficacy of different metalaxyl formulation along with the plant extracts and isolates of T. viride in controlling Alternaria blight and white rust of mustard caused by Alternaria brassicae and powdery mildew was observed and found that application of garlic extract with the application of mancozeb reduced white rust incidence and number of stag heads per plot and increased the seed yield (Meena et al. 2003).

Prasad and Lallu (2006) evaluated efficacy of different spraying combinations of three fungicides and five plant extracts with mancozeb (0.2%) as fungicidal check against Alternaria blight of mustard. Comparative analysis of various spraying schedules revealed that first spray of carbendazim (0.1%) + mancozeb (0.2%) followed by two sprays of mancozeb (0.2%) at early date of sowing (20th October) was the best combination in reducing the disease severity on leaves (18.7%) and pods (10.4%) and observed increased yield, 1000 seed weight and oil content.

Kumar et al. (2008) concluded that Seed treatment with Trichoderma harzianum and its application along with varmicompost into the field recorded maximum in disease control (62.57%), wide head periphery (55.54 cm) and higher yield (398.53 q/ha) in comparison to other treatments.

Latha et al. 2009 studied antimicrobial activity of plant extracts and induction of systemic resistance in tomato plants by mixtures of PGPR strains and Zimmu leaf extract against Alternaria solani, causal agent of early blight of tomato. In vitro studies with leaf extract of Zimmu (Allium cepa L. x Allium sativum L.) demonstrated the highest inhibition of mycelia growth (87%) of A. solani. Biocontrol agents Pseudomonas fluorescens (Pf1 and Py15) and Bacillus subtilis (Bs16) was also tested alone, together, and in conjunction with the most effective plant extract, Zimmu (Allium cepa L. x Allium sativum L.), in both in vitro and in vivo experiments for control of A. solani. All isolates were found compatible with each other and with Zimmu leaf extract. Among the various bioformulations tested as seed treatment and foliar application, the talc-based formulation of Pf1 + Py15 + Bs16 + Zimmu was evaluated superior in induction of defense enzymes, such as peroxidase (PO) and polyphenol oxidase (PPO) phenylalanine ammonia-lyase (PAL), chitinase and b-1, 3-glucanase and accumulation of phenolics and as well as reduced the early blight disease incidence when compared to other treatments. Their study revealed the probable influence of plant growth promotion and induced systemic resistance (ISR) in enhancing the disease resistance in tomato plants against early blight disease by Zimmu based PGPR mixture.

Rathi and Singh (2010) evaluated the efficacy of different bio-agents, plant extract, and fungicides withdifferent combinations as seed treatment and foliar spray against Alternaria blight and white rust diseases in Indian mustard (Brassica juncea). Seed treatment with Trichoderma harzianum @ 10 g/kg seed followed by foliar spray of Ridomil MZ 72 WP (metalaxyl 8% + mancozeb 64%) @ 2 g/ l water after 50-60 days of sowing, significantly reduced the Alternaria leaf and pod blight and white rust of mustard.

Eco-friendly managements (garlic bulb extract, Trichoderma harzianum as seed treatment alone or in combination with foliar spray bay garlic aqueous extract, T. harzianum and Pseudomonas flurescens) of Alternaria blight of Indian mustard was observed by Meena et al. (2011) and found that treatments do not differ significantly among themselves.

Rashid and Hossain(2011) reported that BAU-Bio-fungicide (2.0%) and Iprodione (0.25%) foliar application showed signi?cant e?ect in reducing Alternaria blight and increased seed yield by 120.45% and 97.73%, respectively over non-sprayed control in radish sowing at 25 October. They also suggested that BAU-Bio-fungicide ranked second in reducing the disease, but ranked one in increasing seed yield.

Materials And Methods

A series of experiments were conducted in the laboratory and research plots of the Plant Pathology Department under Bangabandhu Sheikh Mujibur Rahman Agricultural University. The experimental site is located at the centre of Madhupur Tract (24°09′ N latitude and 90°26′ E longitude) having an elevation of 8.2 m from sea level. The soil type of the experimental field belongs to the Shallow Red Brown Terrace type under Salna Series of Madhupur Tract (Brammer, 1971 and Saheed, 1984) of Agro ecological Zone (AEZ) 28 which is characterized by silty clay with pH value of 6.5. The experimental site is under the sub-tropical climatic zone which is characterized by less rainfall, almost clear sun shine and moderate temperature. The experiments were conducted during 2009-2010.

Collection, Isolation and Preservation of Trichoderma and Alternaria

A total of 13 isolates of Trichoderma sp. were used in the study of which 5 isolates were isolated from soil samples, collected from rhizosphere region of lentil, radish, cauliflower, carrot and chilly field and rest of the isolates were taken from the stock culture of microbiology laboratory of BSMRAU. Trichoderma sp. was isolated following the soil dilution plate technique (Dhingra and Sinclair, 1985). The isolates of Trichoderma sp were identified on thebasis of colony growth and morphological characters following the standard key (Barnett, 1980). The pure culture of Trichoderma harzianum was preserved by using PDA slants at 10 C in refrigerator as stock culture for further use.

Alternaria brassiceae and A. brassicicola were isolated from the Alternaria blight infected leaves and pods of radish plants of BARI and BSMRAU farm. Infected samples were cut into small pieces (5 X 5 mm), dipped in 2% sodium hypochlorite solution for 30s and rinsed with sterilized water 3 times. The plant samples were dried by placing on sterilized tissue paper before transferring to a Petri dish containing Potato dextrose agar (PDA). The petridishes were enfolded with plastic tape and incubated at room temperature for 2 days. The isolates of Alternaria were identified on thebasis of colony growth and conidia characters following the standard key (Barnett, 1980). Most of the identified isolates of Alternaria were found as A. brassiceae and only few were isolates were A. brassicicola. Disease symptoms development was tested on a detached leaf of radish under moist petri-dishes by spraying spore suspension of both the species. The pure culture of Alternaria brassicae isolate AB 2 produced maximum number of spots on the detached radish leaves was preserved for future use.

Experiment 1. Screening of Trichoderma isolates against Alternaria brassiceae.

In vitro tests were conducted to evaluate the antagonistic effect of 13 isolates of Trichoderma harzianum against Alternaria brassiceae isolate AB 2on potato dextrose agar (PDA) mediumfollowing Dual Culture technique (Dhingra and Sinclair, 1985). The medium was prepared by mixing infusion of 200 g potato slices, 20 g dextrose, and 20 g agar per liter of medium. After preparation it was sterilized in an autoclaved at 121°C under 1.1 kg/cm2 pressures for 15 minutes. The medium was poured into petridishes (9 cm) at the rate of 20 ml per plate. Young mycelial discs of Trichoderma isolates of 5 mm in diameter were cut from the peripheries of expanding colonies grown on PDA of three days old culture with a cork borer. Disc of Trichoderma were placed at the edges of each PDA plate and one disc of A. brassiceae was placed in the centre. All plates were incubated in the dark for 25 C until the mycelium of A. brassiceae covered the whole the control. Radial growth of the fungus was measured by averaging the two measurements of diameters taken at right angles for each colony. The plates were arranged in Completely Randomized Design (CRD) with three replications. Antagonistic effect of 13 isolates was recorded on the basis of inhibiting potentiality after seven days of incubation. Inhibition percentage of A. brassiceae was calculated based on the growth of the pathogen on PDA plates following the formula as suggested by Sundar et al. (1995):

% Inhibition of growth = 100


X = Mycelial growth of pathogen in absence of Trichoderma (control),

Y= Mycelial growth of pathogen in presence of Trichoderma.

Table 1. Details of Trichoderma isolates
Isolate Source/ Crop Location Collection time
T -8 Stock culture, PLP lab BSMRAU July, 2009
T -18 Stock culture, PLP lab BSMRAU July, 2009
T -16 Stock culture, PLP lab BSMRAU July, 2009
T -21 Stock culture, PLP lab BSMRAU July, 2009
T -25 Stock culture, PLP lab BSMRAU June, 2009
T -2 Stock culture, PLP lab BSMRAU June, 2009
T -77 Stock culture, PLP lab BSMRAU July, 2009
T -3 Stock culture, PLP lab BSMRAU July, 2009
THI -9 Lentil Field, BSMRAU July, 2009
THI -10 Radish Field, BSMRAU July, 2009
THI -11 Cauliflower Field, BSMRAU July, 2009
THI -12 Carrot Field, BARI June, 2009
THI -13 Chilly Field, BSMRAU July, 2009

Experiment 2. Efficacy of different integrated treatment in controlling Alternaria blight of radish seed crop

An experiment was conducted to study the efficacy of eight different treatments which was tested against Alternaria blight of radish seed crop under field condition. The treatments were

T1= Control 1 (Only healthy seeds)

T2= Control 2 (Healthy seeds + Alternaria inoculation)

T3= Trichoderma treated seed+ Alternaria inoculation

T4= Rovral treated seed+ Alternaria inoculation

T5= Trichoderma treated seed+ Alternaria inoculation + Rovral (foliar spray)

T6= Neem leaf extract (foliar spray) + Alternaria sp inoculation

T7= Rovral treated seed+ Alternaria inoculation + Trichoderma sp. (foliar spray+ soil application)

T8= Neem leaf extract (foliar spray) + Alternaria sp inoculation + Trichoderma sp. (foliar spray + soil application) + Rovral (foliar spray)

Collection of Seeds

Seed samples of Radish (Raphanus sativus L.) variety BARI mula-1 was collected from Bangladesh Agricultural Research Institute (BARI).

Land preparation

The experimental field was ploughed properly with a tractor and crop residues of previous crop were removed. All preparations were done to get good tilth. Standard doses of manure and fertilizers were used (Anon. 2004) as follows: Cow dung @ 10mt/ha, Urea @ 350 kg/ha, TSP @ 250kg/ha and MP (Murate of potash) @ 225kg/ha. The entire quantity of cow dung, TSP and 100 kg MP were applied at final land preparation. Urea and rest of MP were applied at two equal installments as top dress.

Design and layout of the experiment

The experiment was laid out in a Randomized Complete Block Design (RCBD) with four replications. After final preparation the field was divided in 2m X 1.5m unit plots maintaining a distance of 50 cm between two plots. In between blocks 50cm wide drains were made for maintain proper drainage. Seeds were sown on Novermber15, 2009 at the rate of 2.5 kg per hectare.

Intercultural operations

After sowing seeds, the field was watered for seed germination. During growing period weeding, mulching and irrigation were done as and when necessary. Aphid infestation was controlled by spraying Malathion 57 EC @1.5 ml per liter water. Rouging was done 3 times at root maturing stage, stem elongation stage and flowering stage.

Preparation and application of substrate with Trichoderma harzianum isolate T-2 in soil

The sterilized wheat bran substrates were prepared and inoculated with theTrichoderma harzianum isolate T-2 which was found effective to control soil borne pathogens in earlier study. The substrates were allowed to colonize by the antagonist for 21 days. After 21 days the colonized wheat grain substrates were air dried ground with a warring blender and stored in conical flasks at 10 C. Trichoderma colonized substrates were mixed in the field soil one month before seed sowing.

Inoculation of Alternaria brassiceae AB-2

For plant inoculation with A. brassiceae aqueous spore suspensions (5.0 X 105 spores /ml) were prepared from 10 days old pure culture of the pathogen. About 0.1 ml of Tween-20 was added to the spore suspension before spraying the inoculum suspension to prohibit the spores from clumping. Spore suspension was sprayed with micro-sprayer prior to flowering at 50 DAS.

Spraying of Rovral 50% WP

Rovral 50WP @ .20% was applied as foliar spray with a knapsac hand sprayer for 4-times at ten days interval starting from flowering stage. With these treatments Rovral 50WP was also used as a seed treating fungicide at the rate of 0.25%. Seeds were taken on petridish and shaken with fungicides for 15 minutes before sowing in the plots.

Spraying of Neem leaf extract

To prepare neem (Azadiracta indica) leaf extract 100g leaves of neem plant was blended in 100ml tap water in a blender and filtered through 3-ply cheese cloth to remove leaf debris. To prepare suspension 10ml extract of a material was mixed with 90ml water. Neem extract 10% was sprayed 4 times at ten days interval from flowering stage.

Foliar spray of Trichoderma harzianum

Aqueous suspension of Trichoderma harzianum spore (5X 105 spores/ml) was prepared from pure culture of Trichoderma isolate T-2. About 0.1 ml of Tween-20 was added to the spore suspension before spraying the inoculum suspension to prohibit the spores from clumping. Prepared suspensions were sprayed with micro-sprayer 3 times after inoculation of A. brassiceae in the field, at pod forming stage and pod maturing stage. Seed was also treated with Trichoderma @ 10ml spore suspension (5X 105 spores/ml) /kg. Tween-20 was added for better attachment of conidia with seed. Plots under control treatment were sprayed with plain water.

Collection of data

Data on severity of Alternaria blight, yield and yield contributing characters of radish seed were recorded from five selected plants. Severity of Alternaria blight on leaves was recorded as percent diseased leaf area of selected plants after initiation of pod. The disease severity was scored using a modified scale (0-5) suggested by Mian (1995), where 0 = no infection, 1 = 1-10%of total leaf area infected, 2 = 11-20% of total leaf area infected, 3 = 21-30% of total leaf area infected, 4 = 31-40% of total leaf area infected, 5 = more than 40% of total leaf area infected.

Data on severity of Alternaria blight on pod, number of spot per pod and percent disease index (PDI) were recorded following a standard formula (Mian 1995). To count the spots on pod and to calculated PDI, 100 pods were randomly selected from every plot. Severity of Alternaria blight on pod was indexed on a scale of 0-5 (Anon.1997, Mian 1995), where 0= no spot, 1 = 10 % pod area diseased, 2 = 20% pod area diseased, 3 = 30 % pod area diseased, 4 = 40% pod area diseased, 5= more than 40% pod area diseased. Percent disease index (PDI) for Alternaria blight on leaf and pod was calculated according to a standard formula by Mian 1995:


Collection of data on seed yield and yield contributing characters

Pods of radish were harvested at full ripening stage. They were collected separately from each plant and kept them in paper bags. Pods were sun dried and seeds of 100 pods were collected in different envelopes. Seeds were sundried, cleaned and stored for further study. Data on seed yield and some yield contributing parameters such as pod number per plant, pod length, 1000 seed weight, seed yield per plant, seed yield per plot were calculated. Seed yield was recorded after threshing and drying in the sun.

Seed Health test

Seed quality in terms of seed grading, germination and prevalence of A. brassicae and A. brassicicola in seed was determined. Seeds were graded into three grades based on seed size. For grading seeds were passed through 10, 12 and 16 mesh sieves were graded as Grade-1, Grade-2 and Grade-3, respectively. Weight of seeds under each grade was determined.

For seed health and germination test, 200 seeds were taken randomly from each treatment plots. Two hundred seeds were placed on eight replicated plates containing moist blotter paper following little modifications of International rules for seed health testing (ISTA, 1993). Seeds were placed on 3 ply moist blotter paper (whatman No. 1) in sterilized 9 cm petridishes. Twenty five seeds were placed in each Petridis maintain equal distances from seed to seed. Before placing in the petridish, the blotter papers were sterilized in at autoclave for 20 minutes at 121 C in 1.1 kg/cm2 pressure. After plating, the petridises were incubated in the microbiology laboratory of BSMRAU at room temperature (25±40 C). To keep the blotter paper moist, sterile water was added whenever necessary. Seven days after plating, number of seeds germinated and number of seeds yielding A. brassicae and A. brassicicola were counted. Germination and prevalence of the pathogen were expressed in percentage based on total number of seeds plated.

Result and Discussion

Experiment. 1. Screening of Trichoderma harzianum isolates against Alternaria brassiceae

A total of selected 13 isolates of Trichoderma harzianum were tested against Alternaria brassiceae isolate AB-2 on PDA by dual culture technique and the results of the screening experiment are presented in Table 2 & Plate I. All the tested isolates of Trichoderma harzianum showed more than 65% inhibition of the radial growth of the test pathogen Alternaria over the control. Among the tested isolates, T- 2 showed the highest (79.63 %) reduction of the radial growth followed by THI-9 (78.89 %) and T-21 (78.33%) against Alternaria brassiceae isolate AB-2. The lowest radial growth inhibition of Alternaria was observed by isolate THI-13 (69.08 %). Significant reduction of mycelial growth of Alternaria in presence of Trichoderma harzianum were also reported by many other workers (Intana et al.2005, Kumar and Parveen 2002) and the results of the present investigations are in agreement with the above mentioned investigators.

Table 2. Screening of Trichoderma harzianum against Alternaria brassiceae isolate AB-2 in dual culture technique

Trichodema isolates Percent of Inhibition
T -8 72.96cde
T -18 71.11 de
T -16 74.44 cd
T -21 69.08 e
T -25 78.33 ab
T -2 79.63 a
T -77 75.19 bc
T -3 75.93 abc
THI -9 78.89 ab
THI -10 74.07 cd
THI -11 72.22 cde
THI -12 72.59 cde
THI -13 69.44 e
Control (9 cm)

Values within a column with a common letter do not differ significantly (P= 0.05).


Plate I. Growth inhibition of Alternaria brassiceae by T. harzianum isolates T-2 in dual culture technique on PDA.

A. Alternaria brassiceae colony on PDA plate (Control)

B. Growth inhibition of Alternaria brassiceae (centre of the plate) by T. harzianum.

Experiment 2. Integrated management for disease free seed production of radish

Disease Development

Alternaria blight first appeared on leave as characteristics Alternaria spots having concentrated rings (Plate II). After pod formation it appeared on pods as small dark brown to black spots (Plate II). Crush mounts were prepared using spotted tissues of leaves and pods, and observed under compound microscope for confirmation of the causal agent as Alternaria brassiceae (Plate III).

Disease severity

The highest severity of Alternaria blight was observed in Control 2 where only pathogen was inoculated. The lowest severity of Alternaria blight was observed in the treatment T8 where integration of fungicide, antagonist ant plant extract were applied followed by the treatment T7, T4, T5 and T6. Integration of Trichoderma, Rovral 50% WP and neem leaf extract 10% in the treatment T8 was appeared identical with the treatment T 7 where Rovral 50% WP and T. harzianum was applied in case of disease severity of both in the leaf and pod. The highest 72.2% and 75.28% reduction of Alternaria blight in leaf and pod was achieved with treatment T8 while only Trichoderma treated seeds in the treatment T3 and neem leaf extract spray in the treatment T6 were appeared to be identical and most inferior in reducing the disease both in leaf and pod infections. Lesion area per leaf spot was highest in control plots which were observed 13.32 and 13.36 cm2/leaf spot in T1 and T2, respectively. Lesion area per leaf spot was lowest in the treatment T8 (2.247 cm2/ leaf spot). Similarly, numbers of spot/pod and lesion area per pod spot were also significantly higher in control plots in comparison to the treated plots. The results of the study (Table 3 and Figure 1) suggested that Rovral 50% WP alone reduced more than 50% Alternaria blight either applied as seed treating fungicide or applied as spraying fungicide. When Rovral 50% WP was integrated with Trichoderma in the treatment T7, percent reduction of disease severity of Alternaria blight of radish was significantly increased compared to the individual application of fungicide, Trichoderma or neem leaf extract and appeared to be identical in reducing disease severity with the treatment T8.

However, this study suggested that integration of fungicide, biocontrol agent and Neem leaf extract in the treatment T8 was found to be best in controlling Alternaria blight of radish. The results of the present study are in agreement with the recent study of Rashid and Hossain (2011) who observed that BAU-Bio-pesticide (Trichoderma) and Iprodione (0.25%) foliar application showed significant reduction of Alternaria blight and increased seed yield of radish.


Plate II. Alternaria blight symptom in the Leaf (A) and Pod (B) of radish.

Plate III. Conidia of Alternaria brassicae

Table 3. Effect of integrated use of Trichoderma, fungicide andbotanicalon incidence and severity of Alternaria blight of radish seed crop.
Treatments Alternaria blight on leaf Alternaria blight on pod
PDI(%) Lesion area/leaf spot(cm2) Number of spot/pod PDI(%) Lesion area/ spot(cm2)
T1=Control 1 63.40b 13.32a 28.55a 56.80ab 13.83a
T2= Control 2 (pathogen) 69.80a 13.36a 31.83a 62.20a 14.05a
T3= T2+ Trichoderma treated 58.00c 8.882c 17.77bc 55.40b 6.725c
T4= T2+ Rovral treated seed 29.80d 5.960d 15.17c 25.80c 4.358d
T5= T3+ Rovral (foliar spray) 32.60d 2.822ef 9.925d 28.20c 1.957e
T6= T2+ Neem leaf extract 58.80bc 9.858b 19.10b 56.20ab 7.655b
T7= T4+Trichoderma (foliar + soil ) 23.10e 3.155e 9.90d 17.60d 2.070e
T8= T6+ Rovral (Foliar) + Trichoderma (Foliar + Soil) 19.40e 2.247f 9.675d 15.40d 1.720e

Means within same column followed by common letter(s) are not significantly different (P=0.05) by DMRT.

Figure 1. Reduction of Alternaria blight disease severity (PDI) on leaf and pod of radish seed crop over control

Seed yield and yield contributing characters

The effect of fungicide, neem extract and Trichoderma either applied alone on applied in combination on seed yield, seed weight of radish was observed in the Alternaria sprayed field and the results are presented in the Table 4 and Figure 2 and Figure 3. All the treatment increased seed weight and yield significantly over the control 1 and 2. The highest seed weight and yield was observed in the treatment T8 where Trichoderma was used as field spray and soil application, Rovral and neem extract applied as field spray followed by treatment T5 where Rovral treated seeds were used following Rovral applied as field spray. Seed yield and seed weight was almost double in comparison to the control in the treatment T8 and T5. Seed yield and seed weight in the treatment were identical and significantly superior in comparison to the other treatments. In response to seed yield treatment T7 was inferior to the treatment T8 and T5 but significantly superior to the all other treatments. Trichoderma treated seeds used in the treatmentT3and Rovral treated seeds used in the treatment T4 were found identical in increasing seed weight and seed yield of radish crop. Neem extract spray applied in the treatment T6 was very effective in increasing seed yield and seed weight and significantly superior to the T3 and T4 but inferior to T7. The lowest seed yield recorded in the control treatment T2 where pathogen was sprayed without any other control measures but identical with the control treatment T1 where only natural condition was maintained and untreated healthy seeds were sown without pathogen and application of any other treatments.

Results presented in Table 3 and Table 4 showed that PDI was negatively correlated with seed yield. Their relationship was described by regression equation Y = -3.001X + 304.5 where Y= seed yield per plot and X= PDI (Figure 3). The value of R² was 0.538, which indicates that the reduction of seed yield was attributed to the severity of Alternaria blight by 53.8%.

Effect of integrated treatment on seed grade and seed health

Produced seeds in the integrated management experiment were graded into three categories: Grade-1, 2 and 3 based on the seed size arrested in the mesh 10, 12 and 16, respectively. The highest percent grade 1 seeds were produced by the treatment T8 followed by the treatment T5. The seed yield under grade 1, 2 and 3 were ranged from 5.20-18.69, 61.23-67.38 and 16-29.96%, respectively (Table 5). More than 25% seed yielded was categorized as grade 3 in the treatment T3, T2 and T1. The maximum of 87% germination of seed under grade-1 was obtained when radish seeds were yielded in the treatment T8 (Table 6). Its effect on germination was significantly higher as compared to control. Seed germination was higher in the treatment T8 followed by T7 and T5 in case of all the grades. Significantly reduced and identical germination was observed in the seeds yielded from control treatments T1 and T2. No significant increase in seed germination compared to the control treatments T1 and T2 in three grades of seeds produced from the treatment T6 where neem extract was sprayed in the Alternaria sprayed radish crop.

The prevalence of Alternaria sp. associated with harvested seeds under different treatments was tests in the blotter method. The results of the seed health tests are presented in the Table 7. Significantly the highest percent Alternaria was isolated from the seeds produced from the Alternaria sprayed control treatments T2 followed by the control treatment 1 at the three grade of seeds. Significantly the lowest 9.00% Alternaria sp. was isolated from the grade 1 seeds of the treatment T8 and T5. The prevalence of Alternaria in the grade 2 and 3 seeds of the T8 were also significantly lower in comparison to the other treatments. Prevalence of Alternaria in the seeds yielded from neem extract sprayed plots in the treatment T6 was significantly higher in comparison to all the treatments except the control treatments T1 and T2. Significantly the highest 46.33% seeds yielding Alternaria from the grade 3 seeds of the control T2 treatment and identical with the grade 3 seeds of the control treatment T1. Data shown in Table 5 and 6 revealed that seed germination under different grades was also negatively correlated with prevalence of Alternaria sp. (Fig. 4-6).

The results of the present study demonstrated that, integrated use of Trichoderma harzianum, Neem extracts and Rovral was appeared to be the best the best management package not only for controlling Alternaria blight disease of radish but also accelerated the seed weight and seed yield. Health of seeds under Grades-1, 2 and 3 were also improved significantly.

The findings of the current study are in agreement with several investigators who observed that integration of Trichoderma was superior in the management of Alternaria blight of crucifers as well as increased seed yield and improved seed quality (Meena et al. 2003; Kumar et al. 2008; Rathi and Singh, 2010 and Rashid and Hossain, 2011).

Table 4. Effect of integration of Trichoderma, fungicide andbotanicalon seed yield affected by Alternaria blight of radish seed crop.

Treatments Pod length (cm) Pod no./Plant 1000 seed wt.(g) Seed Yield/

plot (g)

Seed yield/ ha