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Research Article
Impact of Fungicides and Biocontrol Agents in Managing Peduncle Blight of Tuberose Caused by Lasiodiplodia theobromae (Pat.) Griffon and Maubl

D. Durgadevi, A. Sankaralingam and S. Prabhu

Peduncle blight, hitherto an unknown disease, was found to be a major limiting factor to the cultivation of tuberose, as the disease incidence was noticed up to 42.60% in pockets of Madurai district. Though, Lasiodiplodia theobromae is an ubiquitous pathogen, its occurrence on tuberose is a new record. The fungus induced confounding symptoms which included blossom blight, peduncle blight and leaf blight at tips as well. The causal agent of the disease was identified as Lasiodiplodia theobromae. The efficacy of fungicides and biocontrol agents effective in in vitro was evaluated in pot culture experiment to manage peduncle blight of tuberose. Foliar application of carbendazim 0.1% at 60, 90 and 110 Days After Planting (DAP) was found to be highly effective in reducing the disease incidence up to 95.50%. Among the bioconrol agents, bulb treatment at 10 g kg-1 followed by three foliar sprays at 0.5% on 60, 90 and 110 DAP using the combination of Tv1, Pf1 and Bs10 was equally effective as that of Pf1 and Bs10 with 65.68 and 64.45% disease reduction, respectively.

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D. Durgadevi, A. Sankaralingam and S. Prabhu, 2014. Impact of Fungicides and Biocontrol Agents in Managing Peduncle Blight of Tuberose Caused by Lasiodiplodia theobromae (Pat.) Griffon and Maubl. Plant Pathology Journal, 13: 203-207.

DOI: 10.3923/ppj.2014.203.207

Received: March 26, 2014; Accepted: July 18, 2014; Published: August 30, 2014


Tuberose (Polianthes tuberosa Linn.) is one of the most important ornamental plants which is extensively cultivated in many sub-tropical and tropical areas of the world (Biswas et al., 2002). Tuberose is commercially cultivated for cut and loose flower trade and also for the extraction of its highly valued natural flower oil. Its blooms, besides cutflower, are extensively used in preparation of garlands, bouquets and floral ornaments for bridal make up. Apart from domestic consumption, cut spikes of tuberose have a good export potential. Diseases appear to be the major constraints to the production of tuberose. Peduncle blight, hitherto an unknown disease, was found to be a limiting factor in the cultivation of tuberose. The causal organism Lasidiplodia theobromae was found to be associated with this disease, producing blossom blight, peduncle blight and blighting of leaf tips (Durgadevi and Sankaralingam, 2012). This fungus has a wide host range and is found throughout the tropics and subtropics (Punithalingam, 1980). The present study was undertaken to manage peduncle blight of tuberose using fungicides.


Isolation of pathogen: The pathogen causing peduncle blight in tuberose was isolated from the samples by tissue segment method on Potato Dextrose Agar (PDA) and the fungus was purified by single spore isolation and maintained on PDA. The causal organism was identified based on spore morphology and confirmed further (ID.NO. 6751/11) by Indian Type Culture Collection Centre (ITCC) of Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi.

Pathogenicity in glasshouse: The pathogenicity of the fungus was confirmed by Koch’s postulates using five numbers of four-month-old healthy plants. Plants were inoculated by making a vertical cut (3 mm) in the peduncle region below the calyx using a sterilized needle and placing a fungal disc over the wound. The inoculated area was covered with moist cotton and wrapped with parafilm. The plants were covered with polythene bags to maintain humidity and monitored for symptom expression. Proper controls were maintained with PDA plugs.

Screening of fungicides in vitro: The relative efficacy of six fungicides viz., Kocide 1011 (35% metallic copper), copper oxychloride, cholorothalonil, carbendazim, azoxystrobin and tebuconazole each at four concentrations (0.05, 0.1, 0.15, 0.2%) was tested against B. theobromae under laboratory condition. The name of fungicides and their chemical names and doses are given in Table 1.

Table 1: Fungicides used for in vitro evaluation against Lasiodiplodia theobromae

For this purpose, poisoned food technique devised by Schmitz (1930) was followed. The requisite quantities of fungicides (0.05, 0.1, 0.15, 0.2%) were incorporated into two percent sterilized unsolidified potato dextrose agar and shaken well make it homogenous, medium was then poured into 90 mm sterilized petri dishes with three replications of each treatment with proper control and allowed to solidify. These dishes were inoculated with 9 mm diameter culture discs of 5 days old culture and these discs were placed in the center of the petri dishes. The petri dishes were incubated at 28±1°C for 5 days. The fungal growth was measured after 5 days and percentage inhibition was calculated.

Isolation of biocontrol agents
Rhizosphere: One isolate of biocontrol agent each in Bacillus spp., fluorescent pseudomonads and Trichoderma spp. was isolated from the rhizosphere of tuberose and the other isolates were obtained from the Depatment of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore. Rhizosphere soil from tuberose was collected and the biocontrol agents were isolated by serial dilution (Pramer and Schmidt, 1956) using Trichoderma selective medium for Trichoderma spp., King’s B medium for fluorescent pseudomonads and Nutrient Agar (NA) for Bacillus spp.

Screening of biocontrol agents in vitro: The antagonistic effect of the biocontrol agents viz., three isolates in each of fluroescent pseudomonads, Bacillus spp. and Trichoderma spp., were tested against L. theobromae by dual culture technique (Dennis and Webster, 1971). Five millilitre diameter mycelial disc of the pathogen was placed at one end of the petri plate containing PDA and the bacterial antagonist was streaked at the opposite end. Inoculation of the pathogen without antagonist served as control and each treatment was replicated three times. When the fungus attained full growth in the control plate, growth of the pathogen and inhibition zone were measured and percentage reduction in growth over control was calculated.

Screening of Trichoderma spp. against L. theobromae: Trichoderma spp. were screened by placing a 5 mm mycelial disc of L. theobromae at one end of the petri dish and placing a 5 mm diameter mycelial disc of Trichoderma at the opposite end. Simultaneous inoculation of pathogen and antagonists were followed. Inoculation of the pathogen without antagonist served as control and each treatment was replicated three times. When the fungus attained full growth in the control plate, growth of the pathogen and inhibition zone were measured and percentage reduction in growth over control was calculated.

Compatibility between biocontrol agents: The compatibility of antagonistic bacteria among themselves was tested by streaking the test bacterium vertically on one side of the NA medium and streaking the other bacterium horizontally. The growth of the bacteria were observed and recorded as positive or negative.

Formulation of biocontrol agents
Bacteria: The isolate of P. fluorescens viz., Pf1 and the isolate of B. subtilis viz., Bs10 that were found to be effective in vitro, were used to prepare talc based formulations. Four hundred millilitre of 72 h old bacterial culture in their respective medium with a population of 9x108 CFU mL-1 were mixed with 1 kg of talc containing 15 g of calcium carbonate and 10 g of CMC. Moisture content of the product was reduced to 20% by shade drying and it was packed in polythene bags for further use (Vidhyasekaran and Muthamilan, 1995).

Fungi: The isolate of Trichoderme viridae viz., Tv1 was cultured in sterilized molasses yeast medium for 10 days. The fungal biomass and broth containing spore concentration of 1x107 CFU mL-1 were mixed with talc at 1:2 ratio. The formulation was air dried and packed in polythene covers (Jeyarajan et al., 1994) and used for further study.

Glasshouse experiment: A pot culture experiment using three biocontrol agents viz., TV1, Pf1, Bs10, their combinations and the fungicides viz., carbendazim and tebuconazole was laid out with 11 treatments replicated three times in completely randomized design. A single bulb of tuberose was planted in each pot containing sterile potting medium (red soil:sand:manure at 1:1:1 w/w/w). The method of application included Bulb Treatment (BT) and Foliar Spray (FS).

Table 2: Effect of biocontrol agents and fungicides on peduncle blight of tuberose under glasshouse condition
BT: Bulb treatment, FS: Foliar spray at 60, 90 and 110 days after planting

Talc based bioformulations were applied at 10 g kg-1 bulb followed by three foliar sprays at 0.5% on 60, 90 and 110 DAP. The fungicides were applied at 0.1% on 60, 90 and 110 DAP. B. theobromae was inoculated by wound inoculation at the peduncle region in all the treated plants. Plants inoculated with the pathogen alone served as control. Healthy controls were also maintained. Disease incidence was recorded on 120 DAI and percentage disease incidence was calculated (Table 2).


Trichoderma viride (Tv1): BT+FS
Pseudomonas fluorescens (Pf1): BT+FS
Bacillus subtilis (Bs10): BT+FS
Trichoderma viride (Tv1)+Pseudomonas fluorescens (Pf1): BT+FS
Trichoderma viride (Tv1)+B. subtilis (Bs10): BT+FS
P. fluorescens (Pf1) + B. subtilis (Bs10): BT+FS
Trichoderma viride (Tv1)+P. fluorescens (Pf1) +B. subtilis (Bs10): BT+FS
Carbendazim (0.1%): FS
Tebuconazole (0.1%): FS
Pathogen-inoculated control
Healthy control


The efficacy of biocontrol agents and fungicides to manage peduncle blight was evaluated in a pot culture experiment (Fig. 1a, b).

Out of six chemicals screened in vitro systemics, tebuconazole and carbendazim inhibited the fungal growth completely. Both were effective even at 500 ppm. However, azoxystrobin at 500 and 2000 ppm was inhibitory to L. theobromae by 54.44 and 67.74%, respectively. There was a significant interaction between fungicide and concentration. Among the non-systemic fungicides, chlorothalonil was found to be effective against the fungal growth at all the concentrations.

Fig. 1(a-b):
Efficacy of fungicides, 1: Carbendazim, 2: Tebuconazole, 3: Azoxystrobin, 4: Cholorotahlonill, 5: Copper oxy choloride, 6: Kocide and 7: Control on the growth of Lasiodiplodia theobromae on, (a) 500 ppm and (b) 1500 ppm

The inhibition at 500 ppm was 67.00% while, the same at 2000 ppm was 78.56% (Table 3). Tebuconazole and carbendazim were found to be significantly superior compare to control in inhibiting the fungal growth. These observation are similar to the findings of Rakholiya et al. (2009) and Shah and Verma (2009) for B. theobromae.

Among the 11 treatments, carbendazim 0.1% (T8) was found to be highly effective with the least disease incidence of 4.00% which indicated 95.50 reduction over control.

Table 3: Inhibitory effect of various fungicides against the growth of Lasiodiplodia theobromae in bio assay test
Figures in parentheses are square root transformed values, CD (p = 0.05), Fungicide = 0.01, Concentration = 0.84, FungicidexConcentration = 0.03

Table 4: Biocontrol agents used for in vitro evaluation against Lasiodiplodia theobromae

Carbendazim was followed by tebuconazole (T9) wherein disease incidence was 8.00% with a disease reduction of 91.00%. Both the treatments differed significantly from others (Table 4). The results pertaining to relative efficiency of spray fungicides and biocontrol agents in pot culture experiment indicated that all the fungicides and biocontrol agents proved superior to un sprayed check in reducing the peduncle blight infection. However, carbendazim 0.1% proved most effective in reducing the disease intensity followed by tebuconazole. These findings are in agreement with the observation made by Rakholiya et al. (2009). In field experiment, carbendazim was found to be highly effective in reducing the infection of B. theobromae in mango, suppressing dieback and wilt. Carbendazim 0.1% spray was found to protect custard apple against the infection by B. theobromae. Triazoles viz., difenconazole and tebuconazole at 0.1% were observed to reduce the twig infection and bark canker of pear incited by B. theobromae (Shah and Verma, 2009).

Among the biocontrol agents, treatment T7 (Tv1+Pf1+Bs10) recorded 30.50% disease incidence followed by T6 (Pf1 +Bs10) with 31.60% incidence and both the treatments were on par. Bulb treatment and foliar spray with Tv1 (T1) recorded 42.00% disease incidence with the least disease reduction of 54.44%. Though, all the three isolates of Bacillus spp. inhibited the growth of L. theobromae, Bs10 was found to be more effective. Strains of B. subtilis have been reported to be inhibitory to B. theobromae by earlier workers (Okigbo, 2005; Swain and Ray, 2009; Swain et al., 2008). The antagonistic activity of the three isolates of P. fluorescens against L. theobromae was found to vary and two isolates viz., Pf1 and FP7 were highly inhibitory to the pathogen. The isolate Pf1 of P. fluorescens was the most effective bacterial biocontrol agent which inhibited the growth of B. theobromae up to 84.8% (Govindaiah et al., 2003; Sharma et al., 2009). Isolates of T. viride were highly inhibitory to L. theobromae. T. harzianum and T. atroviridae have been exploited in the management of fruit rots caused by B. theobromae (Kexiang et al., 2002). Pramod et al. (2007) observed that Trichoderma spp. was found to be effective against post-harvest rot of papaya caused by B. theobromae.


The current study concludes that fungicide like carbendazim and tebuconazole were found to be effective against peduncle blight disease of tuberose.

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