Aspergillus niger - Springer Link

BioControl (2006) 51:229–243 DOI 10.1007/s10526-005-1035-1

Ó Springer 2006

The ability of plant compost leachates to control black mold (Aspergillus niger) and to induce the accumulation of antifungal compounds in onion following seed treatment NURAY O¨ZER* and NAGEHAN DESEN KOŸCU¨ Department of Plant Protection, Faculty of Agriculture, Trakya University, Tekirdag˘, 59030, Turkey *Author for correspondence: e-mail: [email protected] Received 24 May 2004; accepted in revised form 18 July 2005

Abstract. Onion seeds treated with leachates of composts prepared from alfalfa and sunflower stalks, at the dosages of 10% and 20% respectively, were inoculated with Aspergillus niger van Tieghem, causal agent of onion black mold disease. The ability of the leachates to induce the production of antifungal compounds and to control black mold were tested at seedling and set stages. Leachates from both composts were able to reduce disease incidence in sets, but not disease severity in onion seedlings. Extracts from treated seedlings and sets were fractionated by thin layer chromatography for their content of antifungal compounds. There were no significant differences between the fractions of alfalfa and sunflower compost leachates in the inhibition of the mycelium growth of A. niger, with the exception of one fraction. The presence of fluorescent pseudomonads and Pantoae agglomerans [synonym: Erwinia herbicola (Lo¨hnis)] bacteria was determined in both leachates. The population of P. agglomerans was higher in the sunflower compost leachate compared to the alfalfa leachate. The tested strains of both bacteria were able to inhibit mycelium growth of the fungal pathogen in agar tests. This study suggests the possible role of beneficial bacteria in the induction of antifungal compounds in onion against A. niger during seedling and set stages. Key words: antifungal compounds, black mold (Aspergillus niger), compost leachate, onion (Allium cepa L.)

Introduction Plants, attacked by pathogens, may exhibit several biochemical defense responses (Goodman et al., 1986) such as enzyme synthesis (Stahmann and Demorest, 1973) and accumulation of antifungal compounds (Nicholson and Hammerschmidt, 1992; Bennett and Wallsgrove, 1994; Hunt et al., 1997). Water-soluble phenols and flavones are important antifungal compounds expressed in red or yellow

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pigmented onion bulb scales (Link and Walker, 1933). These compounds appear to constitute an important resistance factor preventing spore germination and the penetration of potential fungal pathogens. Enzyme syntheses and accumulation of phenolic compounds occur as the components of resistance against basal rot and black mold during different growth stages of onion (O¨zer et al., 1999a, b, 2003, 2004; O¨zer and Koÿcu¨, 2004). Black mold caused by Aspergillus niger Van Tieghem, which is transmitted by contaminated seeds or soil, usually starts the infection at the germination stage of onion seeds and may continue until storage and during storage (Hayden and Maude, 1992; Hayden et al.,1994a; Koÿcu¨ and O¨zer, 1997; Sirois et al., 1998). The use of chemicals and resistant cultivars do not provide sufficient control (Hayden et al., 1994b; O¨zer, 1998; O¨zer and Koÿcu¨, 1998; Sinclair and Letham, 2001). As an alternative to chemical control, compost extracts have been used as sprays to control foliar diseases of plants (Weltzien and Ketterer, 1986; Weltzien, 1992; Elad and Shtienberg, 1994; McQuilken et al., 1994; Yohalem et al., 1994). Recently, the mechanism of action of extracts, obtained from mature composts, which control the foliar diseases has been attributed to systemic acquired resistance (SAR) (Cronin et al., 1996; Zhang et al., 1998; Oostendorp et al., 2001). Previously, it was demonstrated that seed treatments with leachates from the composted stalks of alfalfa and sunflower inhibited the production of pectolytic enzymes and isoenzymes of A. niger in onion (O¨zer et al., 2002). To our knowledge there are no publications on induced resistance to seed-borne diseases in any plant using seed treatments with compost leachates. The objectives of this study were (a) to investigate the effect of seed treatments with leachates from alfalfa and sunflower stalk composts on the accumulation of antifungal compounds during onion seedling and set stages upon infection by A. niger, and (b) to evaluate the toxicity of the extracted fractions on the growth of A. niger.

Materials and Methods Plant source The stalks of sunflower (Helianthus annuus L.) and alfalfa (Medicago sativa L.) were used as compost materials. The stalks were collected from experimental plots of the Faculty of Agriculture (Tekirdag˘/Turkey) and were air dried at room temperature. Onion (Allium cepa L.)

EFFECT OF COMPOST LEACHATE ON ONION BLACK MOLD

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cultivar Kantartopu, which is susceptible to A. niger (O¨zer, 1998), were used in the experiments as the host plant. Culture of the fungus Aspergillus niger isolate (An6) obtained from naturally infested onion seed was used in the experiment (Koÿcu¨ and O¨zer, 1997). This isolate was found to be the most aggressive isolate by seed inoculation in a previous study (O¨zer and Koÿcu¨, 1997). Inoculum was prepared by growing the fungus on potato dextrose agar (PDA) (Oxoid, Unipath Ltd., Basingstoke, England) at 30 °C for 5 days. A spore suspension containing 1107 spores/ml was prepared from the fungal culture and used as inoculum for seed inoculation. Preparation of leachates from composts The stalks of alfalfa and sunflower were placed in separate containers, for the preparation of the compost. Tap water was added to adjust the moisture level to 50% on a dry weight basis. Both containers were turned upside down every two weeks, and maintained at 20±2 °C for four months. After this period, leached liquids were collected and used as stock solution of the leachate obtained from composts. The stock leachates obtained from composted materials were filtered through cheesecloth and centrifuged at 3000 rpm for 5 min. The supernatants from the leachates of alfalfa and sunflower composts were used at 10% and 20% (v/v) dosages, respectively, diluting in distilled water. These dosages proved to have an inhibitory effect on the activity of a pectolytic enzyme of A. niger (O¨zer et al., 2002). Bacterial content of compost leachates Bacterial strains were isolated and their populations quantified on nutrient broth agar (Oxoid, Unipath Ltd., Basingstoke, England) from a 10)6 dilution of each leachate. A 0.1 ml aliquot of diluted suspensions was pipetted onto four replicate plates and were incubated for 48 h at 24 °C. Colonies were enumerated and populations expressed as colony forming units (cfu) per ml of compost leachate. Bacterial colonies were identified by Prof. Dr. H. O¨zaktan (Department of Plant Protection, Faculty of Agriculture, Aegean University) on King’s B medium (KB) (King et al., 1954) and modified Saccharose Nutrient Agar (SNA) (Miller and Schroth, 1972).

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The effect of bacterial strains on the mycelium growth of A. niger Two strains of fluorescent pseudomonads (SFPs-4 and AFPs-7) and two strains of Pantoae agglomerans (SPa-2 and APa-4) were tested on KB agar plates for positive or negative growth effects on A. niger. The bacterial strains were cultured on KB agar plates for 24 h at 28 °C; the isolate (An6) of A. niger (described above) grew on PDA for 5 days at 30 °C. Three agar disks (7 mm diameter), cut from each bacterial plate, were transferred to the edges of a KB plate, along with a disk of A. niger in the middle of the plate. Each combination of the pathogen and the bacteria in a Petri dish was repeated three times and incubated in the dark at 28 °C until the pathogen grew to the edge of the Petri dish. The mean width of the inhibition zone was measured, i.e. from the edge of the bacterial colony to the nearest sporulation of A. niger. Seed inoculation and leachate treatment The onion seeds were surface-sterilized by immersing them in a 1% solution of sodium hypochlorite for 5 min, rinsed in sterile distilled water, and air-dried on sterile filter paper. The seeds were treated by dipping and shaking them for 30 min in the leachates. The leachate treated seeds were inoculated by soaking in the spore suspension of A. niger for 12 h (Hayden et al., 1994a) and then dried on sterile filter paper. Seeds, treated with sterile distilled water and then inoculated with the pathogen, served as the control. Sixteen replicates, each containing 25 seeds per treatment, were placed on sterile filter paper (Blotter method), moistened with sterilized distilled water, in Petri dishes (9 cm in diameter), and incubated in the dark for 7 days. Stereo microscopic visual ratings of disease development on germinating seeds were made after the incubation period. Germinating seeds were rated according to the following 0–3 scale: 0=no damage; 1=root-tip colonization; 2=colonization of entire roots; 3=no germination; seeds completely colonized, as described by O¨zer et al. (2003). The percentage of disease severity (DS) was calculated following the Towsend Heuberger formula (Unterstenho¨fer, 1963): DS ¼

R ratings of each germinating seed 100 Number of total germinating seed rated 3


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Pot experiment Seeds were inoculated and treated with leachates as described previously. For each treatment, five replicates of 20 seeds were sown into 15-cm diameter plastic pots filled with a steam-sterilized soil mixture of field soil, manure and sand, vol/vol (1:1:1). The experiment was conducted in a growth chamber with an average temperature of 18.6 °C, relative humidity of about 60% and photoperiod of approximately 12–14 h day light. Bulbs of 10 plants from each replicate were collected at random during the fourth month of growth, after the development of sets. Each onion set was cut horizontally, and dissected tissues were cultured on PDA to examine the presence of latent infections (O¨zer and Koÿcu¨, 2004). Finally, disease incidence was recorded as the percentage of black molded sets caused by A. niger. Extraction of ultraviolet light absorbing compounds from onion seedlings and sets, and antifungal activity Seedlings and set bulbs developed from the treated seeds were collected separately and homogenized in 2 ml of 95% ethanol per gram of fresh weight. After 24 h extraction at 25 °C in the dark, ethanol extracts were sterilized through a 0.22-lm pore size membrane filter (MilliporeÒ). Similarly, ethanol extracts from non-treated onion seedlings and sets developed from non-treated seeds (in the pot experiment) served as controls. The extracts (80 ll from each sample) were separated by thin-layer chromatography (TLC) and analyzed on silica gel (TLC plates 60 F254, Merck, Darmstadt, Germany), using chloroform:methanol (10:1) as the developing solvent (Sutton and Deverall, 1984). Additionally, ethanol samples of pure 0.1 M catechol (Sigma Chemical Co., St. Louis, Mo., U.S.A.) and 0.1 M protocathequic acid (Sigma Chemical Co., St. Louis, Mo., U.S.A.) were co-chromatographed as standards. The different compounds separated during chromatographic development were visualized by their fluorescence when examined under an ultraviolet fluorescent analysis cabinet with long (365 nm) and short (254 nm) wavelengths after development. The fractions corresponding to different compounds were marked and their retardation factor (Rf) values were calculated using the formula: distance (cm) moved by analyte from origin/distance (cm) moved by solvent front from origin (Wilson, 2000). The separated fractions were isolated by extraction with 0.5 ml ethanol, added to the silica gel scraped from the TLC plate, at a location corresponding to each compound. Silica gel was removed by centrifugation at

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12,000g for 20 min. The spectral study of each band isolated by TLC was carried out in a Shimatzu-1208 UV/VIS spectrophotometer using Spectrum Scanning Program Pack (Shimadzu Corporation, International Marketing Division, Tokyo, Japan). The absorbance spectra of the different compounds were obtained between 200 and 400 nm wavelength and compared for a preliminary characterization of compounds in treated and control seeds and set bulbs. Fractions from TLC plates, dissolved in ethanol, were allowed to dry in a laminar-flow hood and the residues were dissolved in 500 ll of sterile distilled water to determine their effects on radial growth of A. niger. Antifungal activity of each separated fraction was measured as described by Daayf et al. (2000). An agar disk, (3 mm diameter) was placed in the center of a Petri dish containing PDA, and 50 ll of the dissolved fractions were placed 4 cm away from the fungal disk. The Petri dishes were examined daily for mycelium growth. Results were obtained for the fungus when the control culture (0 ll) reached confluence on the Petri dish. Rate of in vitro inhibition in percentage was calculated by the corresponding reduction of fungal radial growth compared to the control (medium with no dissolved fraction). The experiment was repeated three times. Statistical analysis Quantitative data on the bacterial population of compost leachates, the severity or incidence of disease (%) and inhibition of radial growth (%) regarding each fraction were statistically evaluated with analysis of variance (one way ANOVA) procedures of SPSS (Statistical Package for Social Sciences, Inc., 2001, Model 11.0. Chicago). The Tukey–Kramer test was used to compare the means of treatments at P=0.05 level.

Results Bacterial content of compost leachates and in vitro inhibition of the growth of pathogen Two different bacterial populations, fluorescent pseudomonads and P. agglomerans, were found in the compost leachates (Figure 1). The total number of bacteria recovered from leachates of composted sunflower stalks was higher than from composted alfalfa stalks. There was no significant difference in the population of fluorescent


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Figure 1. Content of bacterial populations of fluorescent pseudomonads and Pantoae agglomerans in leachates from composted stalks of alfalfa and sunflower. Bars sharing any letter in common do not differ significantly (P=0.05) according to Tukey–Kramer test.

pseudomonads in the two compost leachates. The leachates from sunflower compost, however, contained a significantly higher population of P. agglomerans compared to the leachate from alfalfa compost. In in vitro tests, the strains of fluorescent pseudomonads caused inhibition zones of 16.7 mm and 11 mm wide (data not shown), and the strains of P. agglomerans caused inhibition zones of 16.7 mm and 17 mm wide in A. niger. Effect of compost leachate on black mold Seed treatments with the leachates from composted stalks of alfalfa and sunflower had a variable effect on the black mold in onion seedlings and sets. While the treatments with compost leachates did not significantly reduce the disease severity on seedlings compared to the control (Figure 2) both treatments significantly reduced the incidence of black mold in the sets (Figure 3). The lowest incidence of disease (40%) was obtained with the sunflower compost treatment. Detection of antifungal compounds in onion seedlings using TLC and spectrophotometry Several TLC fractions, corresponding to different compounds absorbing UV light, were obtained from the seedlings raised from seeds inoculated with A. niger after compost leachate treatments (Table 1). The fractions separated by TLC were grouped according to their increasing Rf values ranging from 0.06 to 0.83. All the fractions were

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Figure 2. Disease severity of black mold in onion seedlings following seed treatments with leachates of composted stalks of alfalfa and sunflower on black mold in onion seedlings. Disease severity (%) was recorded on germinating seeds 7 days after inoculation. Control: seedlings raised from inoculated seeds with A. niger only. Bars sharing any letter in common do not differ significantly (P=0.05) according to Tukey–Kramer test.

Figure 3. Incidence of black mold in onion sets following seed treatments with leachates from composted stalks of alfalfa and sunflower on black mold in onion sets. Control: sets raised from inoculated seeds with A. niger only. Bars sharing any letter in common do not differ significantly (P=0.05) according to Tukey–Kramer test.

spectrophotometrically characterized by their maximum absorbance at kmax. The maximum absorbance values (Amax) of compounds induced by treatments varied, indicating differences in concentration. Fraction I, II and III (Rf=0.06, 0.10 and 0.42, respectively) were expressed by seedlings of the seeds inoculated with A. niger. These fractions were not detected in the seeds of the control. When the seeds were treated with alfalfa compost leachate, four bands were detected, including fractions I and III and also two new bands, IV (Rf=0.77) and V (Rf=0.83). On germinating seeds treated with sunflower compost leachate, fractions I, II and III and distinct band IV were observed. The amount of UV absorbing compounds increased in the seedlings, raised from seeds treated with compost leachates as compared to the

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Table 1. Compounds absorbing UV light, separated by TLC from seedlings raised from A. niger-inoculated seeds after treatment with leachates from composted stalks of alfalfa and sunflower Fraction Rfb

Alfalfa

Controla

Sunflower

kmax nmc max. abs.d kmax nm max. abs. kmax nm max. abs. I II III IV V

0.06 0.10 0.42 0.77 0.83

264.0 – 274.0 274.5 228.0

0.051 – 0.028 0.029 0.040

264.5 212.5 222.5 222.5 –

0.035 0.099 0.061 0.067 –

264.5 263.0 279.0 – –

0.027 0.023 0.016 – –

a

Seedlings raised from inoculated seeds with A. niger only. Retardation factor (See Materials and methods). c Maximum wavelength. d Maximum absorbance. b

control. These increases were generally higher in the fractions from the sunflower compost treatment than from the alfalfa compost treatment. The ability of the detected compounds to inhibit radial growth of A. niger was examined (Table 2). Three fractions (I, II and III) tested from germinated seeds treated with sunflower compost leachate, and fraction I from those treated with alfalfa compost, were significantly more fungitoxic to A. niger than the control. Detection of antifungal compounds in onion sets using TLC and spectrophotometry Following compost leachate treatments and seed inoculation with A. niger in pots, different compounds absorbing UV light were found to be present in the ethanolic extracts from treated and untreated control set tissues (Table 3). Rf values of two fractions (II and IV) were similar to those detected during screening at the seedling stage. Two novel fractions, Ia1 (Rf=0.17) and Ia2 (Rf=0.37), were also found during set stage. Fractions II, Ia1 and Ia2 were found to be constitutively expressed from control onion sets. In the onion seedlings raised from seeds treated with compost leachates and inoculated with A. niger, a distinct band IV was also observed. Sets raised from the seeds treated with sunflower compost and inoculated with the pathogen, revealed fraction II, which was not found in the sets raised from those treated with alfalfa compost. The amount of this compound, however, increased as compared to the control.

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Table 2. Inhibition of radial growth (% reduction) of A. niger by different fluorescent fractions from seedlings raised from seeds inoculated and treated with leachates of composted stalks of alfalfa and sunflower Fraction

Alfalfa

Sunflower

Control*

I II III IV V

17.8 a** – 6.7 b 0.5 a 0.5

16.4 22.9 27.5 6.7 –

2.7 b 9.8 b 0.5 b – –

a a a a

*

Seedlings raised from inoculated seeds with A. niger only. Values in the same row with the same letters are not significantly (P=0.05) different according to the Tukey–Kramer test. **

Table 3. Compounds absorbing UV light, separated by TLC from sets raised from A. niger-inoculated seeds after treatment with leachates from composted stalks of alfalfa and sunflower Fraction Rfb

Alfalfa

Controla

Sunflower

kmax nmc max. abs.d kmax nm max. abs. kmax nm max. abs. II Ia1 Ia2 IV

0.10 – 0.17 296.0 0.37 258.0 0.77 296.0

– 0.102 0.048 0.011

257.0 296.0 259.5 296.0

0.309 0.040 0.055 0.021

259.0 296.0 295.5 –

0.123 0.020 0.046 –

a

Sets raised from inoculated seeds with A. niger only. Retardation factor (See Materials and methods). c Maximum wavelength. d Maximum absorbance. b

The compounds tested from onion sets raised from treated seeds with sunflower compost water exerted higher antifungal activity to A. niger than those from the alfalfa compost treatment and the control (Table 4). Distinctive fraction IV from sets after both treatments also had antifungal activity. Fraction II obtained from the sunflower compost treatment was characterized by the highest antifungal activity. Discussion Compost extracts have previously been reported to reduce the severity of foliar diseases such as powdery mildew and downy mildew of


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Table 4. Inhibition of radial growth (% reduction) of A. niger by different fluorescent fractions from sets raised from seeds inoculated and treated with leachates of composted stalks of alfalfa and sunflower Fraction

Alfalfa

Sunflower

II Ia1 Ia2 IV

– 2.0 c 22.2 a 21.3 a

31.5 19.0 23.1 21.7

a** a a a

Control* 18.9 b 7.9 b 16.7 b –

*

Sets raised from inoculated seeds with A. niger only. Values in the same row with the same letters are not significantly (P=0.05) different according to the Tukey–Kramer test. **

grape, gray mold of strawberries and late blight of potato (Elad and Shtienberg, 1994; McQuilken et al., 1994; Yohalem et al., 1994). These treatments, however, were variable in efficacy. Zhang et al. (1998) demonstrated that peat mix extracts applied as a spray did not control bacterial speck on cucumber grown in either mix or soil. Our results based on the seed treatments with the leachates from composted stalks of sunflower and alfalfa exhibited a significant reduction in black mold incidence on onion sets. In contrast, they were not effective in reducing disease severity of seedlings. This may be ascribed to the phenomenon that at this stage, A. niger probably develops saprophytically on the roots without invading the tissue as reported by Hayden and Maude (1992). Our study demonstrated that compost leachates induced UV fluorescent compounds in onion which are involved in the control of A. niger during seedling and set development. Hunt et al. (1997) found that these compounds were involved in induced resistance. Different UV absorbing compounds were expressed in onion seedlings and sets raised from seeds treated with compost leachates. None of these separated fractions coincided with catechol and protocathequic acid which represent important fungitoxic compounds in the dry uninfected bulbs of colored cultivars (Link and Walker, 1933). Novel fractions Ia1 and Ia2 from treated and control sets, which were not found in seedlings, were probably developmentally regulated. The amounts of the UV fluorescent compounds in the fractions from seedlings and sets increased after seed treatment with sunflower compost leachate. Most of the fractions tested from the latter were characterized by high antifungal activity on the growth of A. niger compared with those from the alfalfa compost treatment and control.

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Compost extracts contain microorganisms as well as chemical factors that appear to play a role in efficacy (Weltzien, 1992; Elad and Shtienberg, 1994; Yohalem et al., 1994; Cronin et al., 1996). Fluorescent pseudomonads and P. agglomerans isolated from compost leachates used in this study inhibited the growth of A. niger in vitro. These bacteria have not previously been reported as antagonists of A. niger. According to our results, the leachates from the composts showed differences in content of beneficial bacterial populations. Alfalfa compost leachate contained higher populations of fluorescent pseudomonads compared to the population of P. agglomerans. In the sunflower compost leachate, the populations of fluorescent pseudomonads and P. agglomerans were at the same level, and the population of P. agglomerans was higher in sunflower than in alfalfa compost leachate. Reports indicated that these bacteria can induce resistance against different diseases (Liu et al., 1995a, b; Wei et al., 1996; Ongena et al., 2000; Jeun et al., 2004). In our study, it appeared more likely that the high population of total beneficial bacteria in sunflower compost leachate had an important role for inducing resistance in onion against black mold. Collectively, our data provide evidence that leachate from composted sunflower stalks has the ability to stimulate a chemical response in onion associated with increased protection against black mold caused by A. niger.

Acknowledgements This research was supported by the Scientific Research Fund of Trakya University (TUÄF-342). The authors wish to thank Prof. P. Magro, Department of Plant Protection, Tuscia University, Prof. Gustav HOLZ, Department of Plant Pathology, Stellenbosch University and Prof. Ahmet ÇITIR, Department of Plant Pathology, Trakya University for their help.

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