Evaluation of Alternaria Alternata As Biological Control Agent Of

Full Length Research Paper

Evaluation of Alternaria alternata causing leaf spot of barnyardgrass grown in rice fields

Mohammad Reza Safari Motlagh Department of Plant Pathology, Faculty of Agriculture, Rasht Branch, Islamic Azad University, Rasht, Iran.

Accepted 20 October, 2011

Barnyardgrass (Echinochloa spp.) is considered one of the world worst weeds. Fungal pathogens can be exploited as biological agents for the management of agricultural weeds. In this research, Alternaria alternata was isolated from Echinochloa spp. and studied as a biocontrol agent against this weed in paddy fields. Pathogenicity test of isolates was done in desiccators, and revealed the pathogenicity of this fungus and its ability to cause leaf blight on Echinochloa spp. Inoculation was done using a spore suspension consisting of 105 spore/ml distilled water and 1% Tween-20 at the 2-3 leaf stage. Also, seeds germination inhibition test was done. The results indicated that A. alternata was able to infect Echinochloa spp. in a higher rate compared with rice cultivar. Moreover, the results showed that seed germination of Echinochloa spp. was significantly affected by A. alternata in comparison with controls. Cluster analysis using unweighted pair group method with arithmetic mean (UPGMA) method gave two groups. The different isolates of A. alternata showed different reactions in seeds germination test and pathogenicity test. Hence, A. alternata can be considered as a probable bioherbicide for the control of Echinochloa spp. at early stages of growth of weed.

Key words: Echinochloa spp., Alternaria alternata, fungi, biological control.

INTRODUCTION

Barnyardgrass (Echinochloa spp.) is considered one of Biological weed control is the use of living organisms the world worst weeds; it is widespread in irrigated fields, such as fungi, viruses, bacteria and nematodes to gardens, barnyards and other cultivated soils (Royer and manage weeds. In practice, the database of biocontrol Dickinson, 1999). It reduces crop yields and causes programs implemented worldwide shows that the vast forage crops to fail to establish by removing up to 80% of majority of the >350 herbivores released against 133 the available soil nitrogen (Royer and Dickinson, 1999). plant species are insects (plus mites and several The high levels of nitrates it accumulates can poison pathogens) (Upadhyay and Blackshaw, 2007). The use of livestock (Maun and Barrett, 1986). It acts as an alternate biological control agents for weed control may host for several mosaic virus diseases (Maun and Barrett, complement herbicide use by introducing novel modes of 1986). The Echinochloa spp. are major weeds wherever action to mitigate the development of herbicide resistance rice in grown (Holm et al., 1977). Biological weed control (Charudattan, 1991; Mortensen, 1998). represents an economically attractive and ecologically Fungal pathogens can be exploited as biological agents sound alternative management technique (Upadhyay and for the management of agricultural pests, diseases and Blackshaw, 2007). weeds (Evans, 1999). Alternaria spp. is airborne fungi that are considered to have potential for the biological control of weeds (Ghorbani et al., 2000). A pathogen, Alternaria macrospora was used for *Corresponding author. E-mail: [email protected]. control of spurred anoda (Anoda cristata) (Walker, 1981). Tel: 00989111384168. Fax: 00981314223621. Infection of cocklebur by Alternaria helianthi caused leaf 4482 Afr. J. Microbiol. Res.

blight in greenhouse conditions (Bassi and Quimby, Horsfall-Barratt system were applied for Echinochloa spp. (Zhang et 1985). A spore concentration of 107/ml spores of al., 1996; Bertrand and Gottwald, 1997). Alternaria alternata caused 100% mortality of Amaranthus retroflexus (Ghorbani et al., 2000). A specific (N1 1)  (N2  2) .... (Nt t) Disease rating  host strain of the fungus Alternaria cassiae, produced (N1  N2 .... Nt ) typical lesions and killed the weeds such as sicklepod, showy crotalaria and coffee senna (Walker, 1983). A. Where N is number of leaves in each of rate, t is number of alternata caused leaf blight on gooseweed (Sphenoclea treatments. zeylanica) and all inoculated gooseweed plants were Pathogenicity tests of rice were carried out in desiccator. Two killed (Masangkay et al., 1999). The metabolites of A. Petri dishes were placed on two desiccators (one desiccator as alternata sprayed on weeds caused the main broad leaf control). Ten (10) seeds of rice (Khazar cultivar) were placed in each Petri dish. Then, the seeds were sterilized in water bath at a to become grassy and sedge weeds to hurt, wilt and dry temperature range of 52 to 57°C and cultivated in saturated soil and to death (Qiang et al., 2008). incubated at 25°C. Distilled water was added to Petri dishes. After The major goal of this study was evaluation of the 16-18 days, seedlings containing 2 to 3 foliages were inoculated by reaction of Echinochloa spp., being one of the important suspension of spores (Safari Motlagh and Kaviani, 2008). Other weeds in paddy fields in Guilan province in Iran, to A. conditions including concentration of conidia and evaluation alternata pathogen fungus. systems were similar.

Inhibition of seeds germination tests MATERIALS AND METHODS Seeds of Echinochloa spp. were surface sterilized with 0.2% Collection and culture of fungal isolates sodium hypochlorite solution for 2 min. After washing with distilled water, seeds were placed per 10 cm Petri dishes containing wet Leaves with symptoms of the disease Echinochloa spp. were filter papers. Then inoculation was done. Ten (10) seeds were collected from fields at Guilan province of Iran, cut to appropriate transferred to two Petri dishes containing wet filter papers (one Petri sizes and transferred to the laboratory. Samples were surface dish as control). Then cuts of fungus colonies (A. alternata isolates) sterilized with 0.5% sodium hypochlorite solution, washed by sterile were placed on seeds. This test was done with three replications. distilled water and placed on potato dextrose agar (PDA) in Petri The Petri dishes were incubated at 28°C on a 12 h light/dark dishes. Then, Petri dishes were incubated at 28°C in darkness or photoperiod. Evaluation of disease symptoms was performed 7 light on a 12 h light/dark photoperiod for 6-15 days (Safari Motlagh days after inoculation and number and percent of germinated seeds and Kaviani, 2008). was determined (Zhang and Watson, 1997). Conidia were single-sporulated and then, monoconidial isolates of the recovered fungi were maintained on half-strength PDA slants in test tubes as stock cultures (Zhang et al., 1996), also pure Data analysis colonies were placed onto sterilized filter paper. Then, paper filter pieces were incubated in sterilized vials in freezer at -20°C (Safari Motlagh and Kaviani, 2008). Data analysis was done using NTSYS software.

Study and identification of fungi RESULTS AND DISCUSSION

Fungi which had grown were isolated and koch’s postulates were completed for most sample after each collection. Cultures of these The first symptoms of A. alternata appeared 48 h after fungi were submitted to the Research Plant Pathology Institute of inoculation on barnyardgrass and 24-36 h after Iran, for the confirmation of identification. inoculation on rice. Symptoms on Echinochloa spp. were elongated and brownish spots that gradually increased in the top of the leaves and a white layer appeared on some Pathogenicity tests of the leaves and was produced leaf blight (Figures 1 and Pathogenicity tests of weed were carried out in desiccators. In each 2). On the rice cultivar appeared chlorotic spots with a of two desiccators (one desiccator as control) two Petri dishes were white layer (Figures 3 and 4). placed, each containing 10 germinated seeds of Echinochloa spp.. Based on the sizes and types of the spots appeared on At first, seeds of Echinochloa spp. were placed on moistened filter the rice and Horsfall-Barratt system, cultivar of rice was paper in Petri dishes and incubated at 28°C for 24 h in a germinator less affected by the A. alternata compared with with 12 h light/dark photoperiod. Then, seeds were surface sterilized with 0.2% sodium hypochlorite solution for 2 min. Echinochloa spp., and its disease rating was lower and After washing with distilled water 10 germinated seeds (coleoptile showed much tolerance (Figure 5). and radical just emerged) were planted per 10 cm Petri dishes filled The results showed that seeds germination of with saturated soil (Zhang et al., 1996) and incubated at room Echinochloa spp. affected by A. alternata and significant temperature. Distilled water was added to Petri dishes. Seedlings at difference was observed between treatments and the 2-3 leaf stage were inoculated with 105 conidia per ml of A. alternata isolates. To increase the surface adsorption, 1% Tween- controls (Figures 6, 7 and 8). 20 was applied. Evaluation of disease symptoms was performed 7 According to dendrogram obtained from cluster analysis, days after inoculation. Therefore, standard evaluation system and in disease rating index on the barnyardgrass, A. alternata Motlagh 4483

Figure 1. Disease symptoms on barnyardgrass.

Figure 2. Disease symptoms on barnyardgrass.

Figure 3. Disease symptoms on rice.

4484 Afr. J. Microbiol. Res.

Figure 4. The comparison between symptoms of A. alternata on barnyardgrass (right) and rice (left).

Figure 5. Diagram of the comparison of A. alternata mean disease rating in rice and barnyardgrass.

Figure 6. Diagram of the comparison of mean seeds germination percent in treatment and control of barnyardgrass.

isolates divided into 2 groups. Each group consisted of 2 Based on dendrogram obtained from cluster analysis, isolates. Also, in the first group, 2 isolates had similarity in disease rating index on the rice cultivar, A. alternata coefficient of 0.95 (Figure 9). isolates divided into 2 groups. The first group consisted of Motlagh 4485

Figure 7. The seeds germination in treatments of barnyardgrass.

Figure 8. The seeds germination in controls of barnyardgrass.

3 isolates. The second group consisted of 1 isolate. Also, isolates showed different reactions in seeds germination in the first group, 2 isolates had similarity coefficient of test and pathogenicity test. This subject can be related to 0.95 (Figure 10). more genetic diversity in different isolates of Alternaria According to dendrogram acquired from cluster analysis, spp. (Oliveira et al., 2002). in seed germination inhibition of the barnyardgrass, A. In a study, pathogenicity of A. alternata on Amaranthus alternata isolates, divided into 2 groups. The first group retroflexus, increased with increasing spore concentration consisted of 3 isolates. The second group consisted of 1 and length of dew period that caused 100% mortality of isolate. Also, in the first group, 2 isolates had similarity this weed at the 4-leaf stage (Ghorbani et al., 2000). coefficient of 0.95 (Figure 11). Infection and mortality in older plants (>4- leaf stage) In study of the reaction of Echinochloa spp. to A. was lower (Ghorbani et al., 2000) that correspond with alternata, it was found that the disease rating caused by results of this research. Alternaria spp., are considered to this fungus on Echinochloa spp. was higher than what have potential for the biological control of weeds was observed in the studied rice cultivar. Also, seeds (Ghorbani et al., 2000), but growth stages and germination of barnyardgrass was affected by A. environmental factors are important (Ghorbani et al., alternata and reduced in comparison with controls. 2000; TeBeest, 1991; Shabanna et al., 1995). Therefore, A. alternata is effective in seed and 2-3 leaf A. cassiae controls multiple weed species without stages in control of this weed. damage to crop plants (Walker, 1983). The pathogen Based on cluster analysis, was observed significant produced dark brown lesions 1 to 5 mm in diameter on difference between isolates of A. alternata. The different the leaves and stems within 2 days. The lesions enlarged 4486 Afr. J. Microbiol. Res.

Alt106s

Alt108s-1

Alt134s-2

Alt110s-1

0.35 0.60 0.86 1.11 1.36 Coefficient

Figure 9. UPGMA-dendrogram for A. alternata isolates on Echinochloa (based on disease rating index).

Alt106s

Alt110s-1

Alt108s-1

Alt134s-2

0.00 0.13 0.26 0.39 0.52 Coefficient

Figure 10. UPGMA-dendrogram for A. alternata isolates on rice (based on disease rating index). Motlagh 4487

Alt106s

Alt134s-2

Alt110s-1

Alt108s-1

0.00 0.64 1.27 1.91 2.54 Coefficient

Figure 11. UPGMA-dendrogram for A. alternata isolates on Echinochloa (based on seed germination inhibition).

with time on any remaining plants and produced severe REFERENCES stem cankers and defoliation within 7 days (Walker, Bassi A, Quimby PC (1985). Infection of cocklebur by Alternaria 1983). helianthi in Proc. South.Weed Sci. Soc. 38th Annu. Meeting, South. The metabolites tenuazonic acid, iso-tenuazonic acid Weed Sci. Soc., p. 373. and their salts were isolated from the cultures of A. Bertrand PF, Gottwald TR (1997). Evaluation fungicides for pecan alternata. They cause the weeds to display symptom of disease control. In: Hickey KD(ed) Methods for Evaluating Pesticides for Control of Plant Pathogens. 2rd ed. APS Press, pp. 149-164. the disease and killed the weeds within 5 days. The Charudattan R (1991). The mycoherbicide approach with plant proper adjuvants will improve the weed killing activity pathogens. In: TeBeest DO (ed) Microbial Control of Weeds. (Qiang et al., 2008). They have an inhibitory effect of NewYork, Chapman 8 Hall, pp. 24-57. around 50% at 10-4 mol/L on the seed germination of the Evans HC (1999). Biological control of weed and insect pests using fungal pathogens, with particular reference to SriLanka. Biocontrol parasitical weed Striga hermonthica (Qiang et al., 2008). News Inf., 2: 63-68. The fungus A. helianthi has been shown to control Ghorbani R, Seel W, Litterick A, Leifert C (2000). Evaluation of cocklebur weed (Sanyal et al., 2008). A. helianthi resulted Alternaria alternata for biological control of Amaranthus retroflexus. in significant reduction in fresh weight, plant height and Weed Sci., 48:474-480. Holm LG, Plucknett DL, Pancho JV, Herberger JP (1977). The word’s leaf number of multiple-seeded cocklebur as compared to worst weeds distribution and biology.University of Hawaii, Honolulu, the plants treated without the fungus (Sanyal et al., p. 609. 2008). Masangkay RF, Mabbayad MO, Poulitz TC, Watson AK (1999). Host In conclusion, A. alternata can be used as eventual range of Alternaria alternata f.sp. sphenocleae causing leaf blight Sphenoclea zeylanica. Canadian J. Bot., 77: 103-112. mycoherbicide for control of Echinochloa spp., in 2-3 leaf Maun MA, Barrett SCH (1986). The biology Canadian Weeds. 77. stage in rice fields in north of Iran. Echinochloa crus-galli. Canadian J. Plant Sci., 66: 739-759. Mortensen K (1998). Biological control of weeds using microorganisms. In: Boland GJ, Kuykendall LD(eds) Plant-Microbe Interactions and Biological Control, New York, Marcel Dekker, pp. 223-248. ACKNOWLEDGMENTS Oliveira AMR, Matsumur ATS, Prestes AM, Van Der Sane ST (2002). Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD). Genet. Mol. Res., 1: This experiment was supported by the Islamic Azad 350-358. University, Rasht Branch, Iran. Qiang S, Dong Y, Chen S, Cai J (2008). Biological control of weeds 4488 Afr. J. Microbiol. Res.

using the metabolites of Alternaria alternata. Fig.1.Patents.Com. Walker HL (1983). Control of sicklepod, showy crotalaria , and coffee Royer F, Dickinson R (1999). Weeds of Canada and the Northern senna with a fungal pathogen. United States Patent, 4390360. United States. Edmonton. University of Alberta Press, p. 346. Walker HL (1981). Fusarium lateritium: a pathogen of spurred anoda Safari Motlagh MR, Kaviani B (2008). Characterization of new Bipolaris (Anoda cristata), prickly sida (Sida spinosa) and velvetleaf (Abutilon spp., the causal agent of rice brown spot disease in the north of Iran. theophrasti). Weed Sci., 29: 629-631. Int. J. Agric. Biol., 10: 638-642. Zhang WM, Watson AK (1997). Effect of dew period and temperature Sanyal D, Bhowmik PC, Abbas HK (2008). Effect of surfactants on on the ability of Exserohilum monoceras to cause seedling mortality bioherbicidal activity of Alternaria helianthi on multiple-seeded of Echinochloa species. Plant Dis., 81: 629-634. cocklebur. J. Plant Pathol., 1: 104-108. Zhang WM, Moody K, Watson AK (1996). Responses of Echinochloa Shabanna YM, Charudattan R, Elwakil MA (1995). Identification, species and rice (Oryza sativa) to indigenous pathogenic fungi. Plant pathogenicity and safety of Alternaria eichorniae from Egypt as a Dis., 80: 1053-1058. bioherbicide agent for water hyacinth. Biological Control, 5: 123-135. TeBeest DO (1991). Microbial control of weeds. London, Chapman and Hall. p. 453. Upadhyay MK, Blackshaw RE (2007). Non-chemical weed management, principle, concepts and technology. Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada, p. 239.