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Journal ofAGROBIOLOGY

ORIGINAL ARTICLE

Biological control of Aspergillus niger, the cause of Black-rot disease of Allium cepa L. (onion), by Penicillium speciesIbatsam Khokhar, Muhammad Saleem Haider, Irum Mukhtar, Sobia Mushtaq

First Fungal Culture Bank of Pakistan, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

Received: 16th September 2011Revised: 6th January 2012Published online: 31st January 2013

AbstractThe purpose of the study was to explore the control of the onion black rot pathogen, Aspergillus niger, with Penicillium species as biological control agents. Fourteen Penicillium species were isolated from the rhizosphere of different plants. In dual culture agar plate assays, these isolates showed very high antagonistic effects on the growth of A. niger mycelium. Penicillium roqueforti and P. viridicatum greatly inhibited the growth of A. niger by 66% and 60%, respectively, followed by P. bilaii (57%) and P. olsonii (53%). However, it was also observed that the Penicillium species completely overgrew the A. niger colony. The study revealed that some species of the genus Penicillium possessed a high antagonistic effect on the onion black rot pathogen.

Key words: Penicillium species; Aspergillus niger; antagonistic; biocontrol; black rot; onion

J Agrobiol 29(1): 23–28, 2012DOI 10.2478/v10146-012-0003-5

ISSN 1803-4403 (printed)ISSN 1804-2686 (on-line)

http://joa.zf.jcu.cz; http://versita.com/science/agriculture/joa

Ibatsam Khokhar, First Fungal Culture Bank of Pakistan, Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan

ibatsamk@yahoo.com

INTRODUCTION

Allium cepa L. (onion) is an important bulb vegetable crop, commercially grown in many countries of the world including Pakistan (Özer and Köycü 2004). Diseases and deterioration by environmental conditions often occur and result in serious economic losses to this crop (Bondad-

Reantaso et al. 2005). Black-rot of onion caused by Aspergillus niger van Tieghem produces extensive losses under storage conditions (Thamizharasi and Narasimham, 1992).

Plant pathogens cause great losses to agricultural crops and thus threaten food resources all over the world (Baniasadi et al. 2009). As a strong pathogen, Aspergillius niger can cause the rotting of numerous fruits and vegetables (Leong et al. 2004, Diedhiou et al. 2007, Fatima et al. 2009, Mathew 2010). Several chemical and biological methods are used to control onion diseases. However, biological control in a combination of several modes of action viz. competition, restraint of pathogen enzymes, and induced resistance, is effective in controlling

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Journal of Agrobiology, 29(1): 23–28, 2012

diseases (Cook et al. 1996, Haran et al. 1996, Elad 2000, El Hassni et al. 2007). It has already become evident that there is a considerable potential in this for organism production and an exciting promise in its use for biological control, (Pandya and Saraf 2010).

The species of the genus Penicillium are commonly found in soil and some species produce harmful metabolites, for example mycotoxins (Khan et al. 2008). Biological mechanisms have been implicated in disease suppression in some plants by the species Penicillium (Jackson et al. 1994, De Cal et al. 1995, Park et al. 2002, Larena et al. 2003, Ma et al. 2008). Therefore, the present study was carried out to evaluate the feasibility of Penicillium species for their biocontrol potential against A. niger, the cause of black-rot disease of onion.

MATERIALS AND METHODS

Isolation of A. nigerInfected bulbs of onion were collected from a vegetable market in the district of Lahore, Punjab, Pakistan, and carried to the laboratory in dry sterilized bags. The diseased portions of the bulb were cut into small pieces, the surface was sterilized by immersion in 1% sodium hypochlorite solution for one minute, rinsed thrice with sterilized water and dried on sterilized filter paper. Sterilized bulb pieces were placed onto

the 2% MEA (malt extract agar) medium in Petri plates of 9 cm diameter and incubated at 25 °C for 7 days. Pure cultures of A. niger were isolated and maintained on 1% MEA medium for further studies.

Isolation of Penicillium species from soilSoil samples were collected from the rhizospheres of various plants from several sites (Table 1) of the Punjab province (Pakistan). Samples were randomly collected from the depth of 8–12 cm below the soil surface by using an open-end soil borer (20 cm in depth, 2.5 cm in diameter) as described by Lee and Hwang (2002). Samples were air-dried at room temperature for 7–10 days and then passed through a 0.8 mm mesh sieve. They were then stored in polyethylene bags at room temperature before the use. An air-dried soil sample (10 g) was mixed with sterile distilled water (100 ml), shaken vigorously for 15 min and then allowed to settle for 20 min. For serial dilution, 1 ml of soil suspensions was transferred to 9 ml of sterile distilled water (dilution 10–1) and subsequently diluted to 10–2, 10–3, 10–4, 10–5 and 10–6. For fungal isolation, an aliquot of 10–4 soil dilution (0.5 ml) was inoculated onto 2% MEA medium and spread evenly. Three replicates were considered. Plates were incubated at 20 °C for up to 7 days. Penicillium colonies were observed under stereo microscope and were isolated on 2% MEA, incubated at 20 °C for purification.

Table 1. List of Penicillium species isolated from rhizospheric soil samples of various plants

Sr. No Penicillium species Rhizosphere of

1. P. bilaii Dalbergia sisso

2. P. brevicompactum Eucalyptus globulus

3. P. dendriticum Hibiscus esculenta

4. P. duclauxii Psidium guajava

5. P. implicatum Punica granatum

6. P. islandicum Citrus limonia

7. P. italicum Dalbergia sisso

8. P. janczewskii Pennisetum americanum

9. P. olsonii Psidium guajava

10. P. oxalicum Citrus limonia

11. P. roqueforti Eucalyptus globulus

12. P. spinulosum Punica granatum

13. P. variabile Hibiscus esculenta

14. P. viridicatum Citrus limonia

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Identification of fungiThe isolated fungi were identified to the genus and species level on the basis of macro- and micro-morphological characteristics using the most updated keys for identifications (Raper and Fennell 1965, Pitt 1979, Domsch et al. 1980).

Dual culture assayA 10 mm disc of the pathogen with similar size of each Penicillium species was taken from the edge of a 5-day-old pure culture using a cork borer and plated 20 mm apart respectively on 2% MEA medium. The control plates were plated with the pathogen and antagonists separately. Plates were incubated at 25 °C for 14 days. Antifungal activity was indicated as mycelial growth of A. niger isolate was prohibited in the direction of active Penicillium isolates. The growth inhibition was calculated by using the formula:

C - TGrowth inhibition (%) = ─── × 100

CWhere C = growth in control; T = growth in the treated variant.

Statistical analysisThe experiment was carried out using a completely randomized design. Standard errors of means of three replicates were computed using computer software Microsoft Excel. All the data was subjected to analysis of variance followed by mean separation through Duncan’s Multiple Range Test (Steel and Torrie 1980) using the computer software COSTAT.

RESULTS AND DISCUSSION

Fourteen Penicillium species have been isolated from rhizospheres of various plants, viz. P. bilaii (Dalbergia sisso), P. brevicompactum (Eucalyptus globulus), P. dendriticum (Hibiscus esculenta), P. duclauxii (Psidium guajava), P. implicatum (Punica granatum), P. islandicum (Citrus limonia), P. italicum (Dalbergia sisso), P. janczewskii (Pennisetum americanum), P. olsonii (Psidium guajava), P. oxalicum (Citrus limonia), P. roqueforti (Eucalyptus globulus), P. spinulosum (Punica granatum), P. variabile (Hibiscus esculenta) and P. viridicatum (Citrus

0

10

20

30

40

50

60

70

80

Inhi

bitio

n %

age

1. P. bilaii 2. P. brevicompactum 3. P. dendriticum 4. P. duclauxii 5. P. implicatum

6. P. islandicum 7. P. italicum 8. P. janczewskii 9. P. olsonii 10. P. oxalicum

11. P. roquefortii 12. P. spinulosum 13. P. variabile 14. P. viridicatum

hifgh

b

de

ghi

d

c

bc

hi

c

j

ef efg

ij

1 2 3 4 5 6 7 8 9 10 11 12 13 14

 Fig. 1. Effect of Penicillium spp. on growth inhibition of A. niger. Bars topped by different letters are significantly different at P<0.05.

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Journal of Agrobiology, 29(1): 23–28, 2012

limonia). The results showed that Penicillium species can restrict growth of A. niger. The mycelial growth inhibition (%) of A. niger and the 14 Penicillium species has been observed (Fig. 1). A minimum growth inhibition of 12% and 18% of A. niger colony was observed by P. dendriticum and P. janczewskii, respectively. Penicillium roqueforti and P. viridicatum inhibited the growth of A. niger maximally, by 66% and 60%, respectively, followed by P. bilaii (57%) and P. olsonii (53%).

A significant interaction was exhibited by a Penicillium species (a member of the subgenus biverticillium which could not be identified) in which the growth of A. niger was much affected by hyphal interference (Fig. 2). Microscopic observation revealed that Penicillium species overgrew A. niger. When A. niger came in contact with hypha of Penicillium species, it did not show any clear pattern of hyphal interaction but

the antagonistic fungus grew over the colony of A. niger and did not completely inhibit its growth (Fig. 3).

In the present study, antagonistic effects of Penicillium spp. indicated their importance as a possible biocontrol agent. Only P. roqueforti and P. viridicatum showed appreciable reduction in the growth of A. niger. P. dendriticum was the least effective. Microorganisms as biological control agents have high potential to control plant pathogens with no effect on the environment and other non-target organisms. In the present scenario numerous reports are available on the potential use of biocontrol agents as replacements of agrochemicals (e.g., Gomathi and Ambikapathy 2011).

From the observation of mycelium, contact Penicillium species caused damage to A. niger by coagulating its cytoplasm and also caused lysis and tip burst of the pathogen. Results

 

Fig. 2. Interaction of Aspergillus niger with different Penicillium species. A, Aspergillus niger; B, Penicillium bilaii; C, P. brevicompactum; D, P. dendriticum; E, P. duclauxii; F, P. implicatum; G, P. islandicum; H, P. ita-licum; I, P. janczewskii; J, P. oxalicum; K, P. roqueforti; L, P. spinulosum; M, P. variabile; N, Penicillium sp.

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Journal of Agrobiology, 29(1): 23–28, 2012

showed that some Penicillium species completely antagonized A. niger by overgrowth mechanism. This could be a result of antagonism due to parasitism or antibiosis as lytic activity (Gomathi and Ambikapathy 2011). In case of P. variabile, P. dendriticum and P. duclauxii zone of inhibition was clearly observed, whereas other Penicillium species and P. oxalicum showed mutual intermingling growth with A. niger. However, the mutually intermingled growth of some Penicillium species with A. niger without any zone of inhibition indicates the failure of the production of antibiotics either by the pathogen or by the antagonist. The formation of a zone of inhibition is an indication for the production of antibiotic substances either by the pathogen against antagonistic fungi or vice versa (Gomathi and Ambikapathy 2011).

This study revealed that some Penicillium species possessed a high antagonistic effect on the pathogen A. niger. An integrated approach of disease management using the interaction of a biological agent is likely to be more attractive to the growers than a chemical approach alone.

 

Fig. 3. (A, B, C and D). Interaction of Penicillium species and Aspergillus niger mycelia under stereoscope

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