Received date: October 18, 2015; Accepted date: November 02, 2015; Published date: November 05, 2015
Citation: Aydi Ben Abdallah R, Jabnoun-Khiareddine H, Mejdoub-Trabelsi B, Daami-Remadi M (2015) Soil-borne and Compost-borne Aspergillus Species for Biologically Controlling Post-harvest Diseases of Potatoes Incited by Fusarium sambucinum and Phytophthora erythroseptica. J Plant Pathol Microbiol 6:313. doi:10.4172/2157-7471.1000313
Copyright: © 2015 Aydi Ben Abdallah R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Nine isolates of Aspergillus spp., isolated from soil and compost were tested in vitro and in vivo for their antifungal activity against Fusarium sambucinum and Phytophthora erythroseptica, the causal agents of the Fusarium dry rot and pink rot of potato tubers. Tested using the dual culture method, the pathogen growth of F. sambucinum and P. erythroseptica was inhibited by 27 to 68% and 16 to 25% by all Aspergillus species, respectively. The highest inhibitory activity against both pathogens was induced by the isolate CH12 of A. niger. A significant reduction of the mycelial growth of both pathogens tested using the inverse double culture method involves the presence of volatile antifungal metabolites. Their effectiveness was also evaluated as tuber treatment prior to inoculation with the pathogens. The highest effectiveness in reducing Fusarium dry rot severity was recorded on tubers treated with the isolate CH12 of A. niger. This study also revealed that the efficacy of Aspergillus spp. as biocontrol agents may be enhanced by varying the timing of their application. In fact, the lesion diameter of dry and pink rots was reduced by 54-70 and 52% with preventive application, respectively. However, this parameter decreased by 21-48 and 47% when the Aspergillus spp. were applied simultaneously with pathogens, respectively. Similarly, diseases’ severity, estimated based on average penetration of F. sambucinum and P. erythroseptica, was reduced by 57-77 and 55% with preventive treatments and by 29-68 and 44% with simultaneous application, respectively. This study reveals that Aspergillus spp., isolated from compost and soil, exhibits an interesting antifungal activity toward F. sambucinum and P. erythroseptica and may represent a potential source of biopesticide. Testing of their culture filtrates, their organic extracts and their toxicity may give additional information on their safe use as biocontrol agents.
Aspergillus spp; Biological control; Dry rot; Fusarium sambucinum; Mycelial growth; Phytophthora erythroseptica; Pink rot
Potato (Solanum tuberosum L.) is one of the most important vegetable crops in the world [1-3]. In Tunisia, it is one of the strategic crops occupying about 16% of all Tunisian cultivated areas . However, potato production is threatened by several fungal diseases resulting in considerable yield losses [5,6]. These diseases can affect potato at any growth stage or even during storage, where potato tubers may exhibit diverse types of rots. Dry rot induced by different Fusarium species, Pythium leak caused by Pythium spp. and pink rot incited by Phytophthora erythroseptica are responsible for important tuber storage losses in the world and in Tunisia [7-13].
Significant losses in storage with estimates of up to 100% have been reported both in developed and developing countries when disease management is neglected . Losses associated with dry rot have been estimated to range from 6 to 25%, and occasionally losses as great as 60% have been reported during long-term storage . The primary sources of inoculum are contaminated or infected seed tubers and infested soil . Tuber infection by F. sambucinum occurs through wounds produced during planting, harvesting or transport. Dry rot causes a dry and crumbly decay with abundant white, yellow or carmine-coloured mycelium depending on Fusarium species . For pink rot, the oomycete, P. erythroseptica infects potato tubers through stolons or lenticels via zoospores and through cracks and cuts made during harvest and handling operations [5,16-17]. The infected tuber flesh becomes soft, spongy and watery with a light brown color. A clear liquid came out from a cut tuber and the surface acquires a pink coloration which turns brown and darkens after a few hours . Thus, the health of seed tubers, the management practices during the growing period, the harvesting and handling practices and the environmental conditions maintained throughout storage are key factors affecting tuber infection by these pathogens .
Currently, the primary control for these diseases in storage facilities includes elimination of infected tubers prior to storage and storage management using forced air ventilation, and controlled temperature and humidity feedback systems . In fact, there is a shortage of postharvest fungicides to completely manage these pathogens . The control of potato dry rot has been achieved, for many years, by postharvest application of thiabendazole, a Benzimidazole fungicide . However, F. sambucinum resistant to this fungicide and other benzimidazole fungicides were discovered in many parts of the world, leading to reduced effectiveness in controlling dry rot . For pink rot, chemical control is limited to a few compounds. In fact, fungicides containing metalaxyl and mefenoxam were used effectively to control pink rot in the 1990's. However, metalaxyl-resistant isolates of P. erythroseptica are now widespread and this may lead to failure of these chemicals to control pink rot . Phosphorous acid and many other disinfectants are currently registered for postharvest management of pink rot. However, use of these products does not completely control storage pathogens .
Furthermore, all commonly grown potato cultivars are susceptible to potato pink and dry rots. Therefore, lack of available post-harvest fungicides and disease-resistant cultivars has prompted the search for new and efficient alternative methods as seed tuber and/or postharvest treatments to reduce incidence and severity of dry and pink rots.
Recent studies indicate that generally recognized as safe (GRAS) compounds and microbial antagonists could eventually be integrated into dry rot management strategies . In fact, many biocontrol agents have been explored to control potato postharvest diseases such as Pseudomonas spp., Enterobacter spp., Trichoderma spp, Aspergillus spp. which reduced the severity of dry and pink rots caused by F. sambucinum and Phytophthora spp., respectively [25-27]. Aspergillus niger, A. flavus, A. terreus, A. fumigatus have been shown to control potato postharvest diseases caused by Pythuim ultimum [28-30], Fusarium sambucinum  and Phytophthora sp. . These Aspergillus species used as biological control agents against many fungal pathogens such as F. oxysporum, Sclerotinia sclerotiorum, Pythium spp. act through mycoparasitism, competition, mycelial lysis and antibiosis via the synthesis of volatile and/or non-volatile metabolites [11,33-34]. Aspergillus species are ubiquitous in most agricultural soils and they generally produce a variety of secondary metabolites exhibiting inhibitory effects on several soil-borne microorganisms . Moreover, many Aspergillus species are considered among the potential thermotolerant antagonists surviving after soil solarisation such as A. terreus, A. ochraceus and A. fumigatus . They are also among the most abundant in composts [37-39] and they play a significant role in the composting process as well as in suppressing the growth of pathogenic fungi [40-42]. In recent studies, many atoxigenic Aspergillus spp. strains, mostly isolated from soil, were used as effective biocontrol agents against Aspergillus toxigenic strains . In fact, naturally occurring populations of atoxigenic strains are considered as potentially important reservoirs for the selection of strongest biocompetitors . Furthermore, Frisvad and Larsen  mentioned that the genus Aspergillus is rich in species and these species are able to produce a large number of extrolites, including secondary metabolites, bioactive peptides/proteins, lectins, enzymes, hydrophobins and aegerolysins.
Therefore, the objective of the present study was to evaluate the in vitro and in vivo antifungal potential of Aspergillus species (A. niger, A. terreus, A. flavus, Aspergillus sp.) isolated from compost, solarised and non-solarised soils to control F. sambucinum and P. erythroseptica. The timing of their application as tuber treatment was also tested to optimize their suppressive effects toward potato Fusarium dry rot and pink rot.
The isolates of F. sambucinum and P. erythroseptica used in this study were obtained from potato tubers showing typical symptoms of dry rot and pink rot, respectively. They were cultured on Potato Dextrose Agar (PDA) and incubated at 25°C for seven days before use.
Four Aspergillus species namely A. niger, A. terreus, A. flavus and Aspergillus sp., isolated from soil or compost, were used in this study (Table 1). They were cultured on PDA medium and incubated at 25°C for seven days before being used in the bioassays.
|Isolate||Species||Origin||Year of isolation|
|CH2||A. terreus||Solarized soil||2006|
|CH8||Aspergillus sp.||Non-solarized soil||2006|
|CH12||A. niger||Non-solarized soil||2006|
Table 1: The soil-borne andcompost-borne Aspergillus species tested against Fusarium sambucinum and Phytophthora erythroseptica and their isolation sources.
Assessment of the in vitro antifungal activity of Aspergillus spp. against Fusarium sambucinum and Phytophthora erythroseptica
The antagonism of Aspergillus spp. isolates against F. sambucinum and P. erythroseptica was tested in vitro using the dual culture method and the inverse double technique on PDA medium. The first technique consists in depositing equidistantly two agar plugs (diameter 6 mm) colonized by the pathogen (removed from a 7-days-old culture at 25°C) or the antagonist (removed from a 7-day-old culture at 25°C) in the same Petri dish containing PDA medium supplemented with streptomycin sulfate (300 mg/L) .
The second method consists in transplanting the antagonist and the pathogen in two separate Petri dish containing PDA medium supplemented with streptomycin sulfate (300 mg/L). Thereafter, a 6 mm agar plug of the antagonist, removed from the margin of an actively growing culture, was placed in the centre of the bottom dish whereas the agar plug (diameter 6 mm) colonized by the pathogen (removed from a 7-days-old culture at 25°C) was placed in the centre of the top Petri dish. Both dishes were sealed with parafilm layers to prevent loss of volatile substances . The two pathogens (F. sambucinum and P. erythroseptica) were thus exposed to the influence of the volatile substances released from isolates of Aspergillus spp. tested. Control cultures are challenged by the pathogen only in the PDA medium without transplanting antagonists, for the two confrontation methods.
The diameter of the pathogen colony (treated and control) was measured after 7 and 4 days of incubation at 20°C for F. sambucinum and P. erythroseptica, respectively. The mycelial growth inhibition percentage was calculated according to the following formula:
I ℅=[(C2-C1)/C2] × 100 with C2: Mean diameter of the control colony and C1: Mean pathogen colony diameter in the presence of the antagonist .
Assessment of the in vivo antifungal activity of Aspergillus spp. against Fusarium sambucinum and Phytophthora erythroseptica
Apparently healthy potato (cv. Spunta) tubers were used in this study. They were washed under running tap water to remove excess soil, dipped into 10% sodium hypochlorite solution for 5 min, rinsed twice with sterile distilled water (5 min each) and air-dried.
Tubers were wounded at two sites along the tuber longitudinal axis by a disinfected Pasteur pipette occasioning wounds of 6 mm in diameter and in depth, which serve as infection sites. Tuber inoculation was made by deposing in each wound an agar plug (6 mm diameter) colonized by the pathogen removed from a 7-day-old culture at 25°C.
Conidial suspensions (106 CFU/mL) of the biocontrol agents, tested individually, were applied by injecting 100 μl in each wound either simultaneously with the pathogen or 24 h before. The positive control was inoculated with the pathogen only whereas the negative control was uninoculated and untreated with any of the antagonists tested.
All tubers were incubated at high relative humidity for 21 days at 20°C and for 7 days at 25°C for F. sambucinum and P. erythroseptica, respectively. After this incubation period, tubers were cut longitudinally via sites of inoculation and lesion diameter, width (l) and depth (p) of the occasioned rot were measured. The penetration of the pathogen was calculated using the following formula:
P (mm)=[(l/2) + (P-6)]/2 .
The rate of reduction for the lesion diameter of dry and pink rots and the penetration of the pathogens were calculated using the following formula:
I ℅=[(C2-C1)/C2] * 100 with C2: lesion diameter of the rot or penetration of the pathogen in the untreated control and C1: lesion diameter of the rot or penetration of the pathogen in the presence of the antagonist .
Data were subjected to a one-way analysis of variance (ANOVA) using SPSS 16.0. For the in vitro tests, each individual treatment was repeated three times and the essays were analyzed in a completely randomized model. The in vivo essay was analyzed in a completely randomized factorial model with two factors (antagonistic treatments and timing of application) and each individual treatment was repeated six times. Means were separated using Student-Newman-Keul’s (SNK) test (at P ≤ 0.05).
Effect of Aspergillus spp. on the mycelial growth of Fusarium sambucinum and Phytophthora erythroseptica
Analysis of variance showed a highly significant (at P ≤ 0.05) inhibitory effect of the antagonists tested on the average colony diameter of F. sambucinum and P. erythroseptica, recorded after 7 and 4 days of incubation, respectively. Tested using the dual culture method, F. sambucinum and P. erythroseptica growth at 20°C were inhibited by 27 to 68% and by 16 to 25% by all Aspergillus isolates, respectively. The highest inhibitory activity against pathogens, as compared to the untreated control, was induced by the isolate CH12 of A. niger (Figures 1 and 2). An important inhibition of F. sambucinum by 37, 32 and 35% was also induced by the isolates CH1 and MC2 of A. niger and MC5 of A. flavus, respectively. The isolates CH2 and MC8 of A. terreus, CH8, CH4 and CH3 of Aspergillus sp. reduced the colony diameter of F. sambucinum by 31, 30, 29, 28 and 27%, respectively. A. niger was also active against P. erythroseptica, causing an inhibition of 24 and 22%, by the isolates MC2 and CH1, respectively. An inhibition of P. erythroseptica mycelial growth by 20 to 21% was achieved using the isolates CH2 of A. terreus, MC5 of A. flavus, and MC8 of A. terreus. The isolates of Aspergillus sp. (CH8, CH4 and CH3) were less active against P. erythroseptica with an inhibition of 19, 18 and 16%, respectively (Figure 1).
Figure 1: Effect of Aspergillus spp. on the mycelial growth of Fusarium sambucinum (a) and Phytophthora erythroseptica (b) noted after 7 and 4 days of incubation at 20°C, respectively, using the dual culture method. CH1, CH12, MC2: A. niger; CH2, MC8: A. terreus; CH3, CH4, CH8: Aspergillus sp.; MC5: A. flavus. Bars affected with the same letter are not significantly different according to Student-Newman-Keul’s test at P ≤ 0.05.
Tested using the inverse double culture method, a significant reduction of F. sambucinum mycelial growth, by 27.84 and 27.45% compared to the untreated control was induced by the isolates CH2 of A. terreus and MC5 of A. flavus, respectively (Figures 3a and 4). All Aspergillus spp. tested had significantly inhibited P. erythroseptica by distance culture method from 9.24 to 23.09% compared to the untreated control. The highest reduction of P. erythroseptica mycelial growth was achieved using the isolates CH1, CH12, MC2 of A. niger and MC5 of A. flavus (Figures 3b and 4). An inhibition varying from 15.70 to 17.78% was caused by Aspergillus sp. isolates (CH3, CH4 and CH8) and MC8 of A. terreus. The isolate CH2 of A. terreus showed the lowest inhibition of P. erythroseptica at distance compared to the untreated control (Figure 3b).
Figure 3: Effect of Aspergillus spp. on the mycelial growth of Fusarium sambucinum (a) and Phytophthora erythroseptica (b) noted after 7 and 4 days of incubation at 20°C, respectively, tested using the inverse double culture method. CH1, CH12, MC2: A. niger; CH2, MC8: A. terreus; CH3, CH4, CH8: Aspergillus sp.; MC5: A. flavus. Bars affected with the same letter are not significantly different according to Student-Newman-Keul’s test at P ≤ 0.05.
Effect of the application timings of Aspergillus spp. on potato dry rot and pink rot severity
Results analyzed by ANOVA revealed a significant variation in Fusarium dry rot severity, recorded after 21 days of incubation at 20°C, depending upon treatments tested, timings of their application and their interaction. The lesion diameter of dry rot was reduced by 54 to 70%, as compared to the untreated control, with preventive application. However, this parameter decrease varied from 21 to 48% only when the Aspergillus spp. were applied simultaneously with the pathogen (Figures 5a and 8). Disease severity, estimated based on average penetration of F. sambucinum, was reduced by 57 to 77% with preventive treatments compared with 29 to 68% obtained with simultaneous application (Figures 5b and 8).
Figure 5: Effect of the application timing of Aspergillus spp.-based treatments on potato tubers on the lesion diameter (a) and the average penetration (b) of Fusarium sambucinum noted after 21 days of incubation at 20°C. CH1, CH12, MC2: A. niger; CH2, MC8: A. terreus; CH3, CH4, CH8: Aspergillus sp.; MC5: A. flavus. NIC: Uninoculated and untreated control; IC: Inoculated and untreated control. LSD (Timings of application × Antagonistic treatment tested): 5.8 mm (a) or 2.28 mm (b) at P ≤ 0.05.
Figure 6: Effect of Aspergillus spp. based-treatments of potato tubers on the lesion diameter (a) and the average penetration of Phytophthora erythroseptica (b), noted after 7 days of incubation at 25°C. CH1, CH12, MC2: A. niger; CH2, MC8: A. terreus; CH3, CH4, CH8: Aspergillus sp.; MC5: A. flavus. NIC: Uninoculated and untreated control; IC: Inoculated and untreated control. Bars affected with the same letter are not significantly different according to Student-Newman-Keul’s test at P ≤ 0.05.
Figure 7: Effect of the application timing of Aspergillus spp.-based treatments on potato tubers on the lesion diameter (a) and the average penetration (b) of Phytophthora erythroseptica, noted after 7 days of incubation at 25°C. Bars affected with the same letter are not significantly different according to Student-Newman-Keul’s test at P ≤ 0.05.
The highest effectiveness of Aspergillus spp. in reducing Fusarium dry rot severity, as compared to the inoculated and untreated control tubers, was recorded on potato tubers treated with the isolate CH12 of A. niger in both application timings. This isolate reduced the lesion diameter by 55% and the average penetration of the pathogen by 73%, as compared to the inoculated and untreated control tubers (Figures 5 and 9).
Analysis of variance showed a significant (at P ≤ 0.05) variation in pink rot severity, recorded after 7 days of incubation at 25°C, depending upon treatments tested and the timings of their application. The interaction between these two factors did not show significant variation on the reduction of pink rot severity. Whatever the timing of their application, a reduction of the lesion diameter of pink rot varied between 83 and 88% with all biological treatments tested compared to the inoculated and untreated control (Figure 6a). All isolates of Aspergillus spp. tested had also limited the average penetration of P. erythroseptica by about 82 to 91% compared to the inoculated and untreated control (Figure 6b).
A significant decrease in pink rot severity was also achieved using the antagonists tested preventively. The lesion diameter of pink rot was reduced by 52% with preventive application as compared to 47% when the Aspergillus spp. were applied simultaneously with the pathogen (Figure 7a and 8). All antagonists tested and applied 24 h prior to inoculation with P. erythroseptica led to 55% decrease in the average penetration of the pathogen compared to 44% obtained with simultaneous application (Figures 7b and 8).
In recent years, intense research efforts have been devoted to the development of antagonistic microorganisms to control potato diseases such as Penicillium, Trichoderma, Aspergillus, Gliocladium, etc [11,48]. In fact, several studies have promoted the antagonistic activity of Aspergillus species which have been identified in compostor residue-amended substrates, in solarised and non-solarised soils, in the rhizosphere, in decaying plant material, in stored grains, etc. Furthermore, some of them are essential component of many compost microbiota [28,49-52]. Moreover, Aspergillus species have been reported as endophytes with antifungal activity [53-55] and able to produce several metabolites such as phenolic and bioactive flavonoid compounds.
In the current study, the use of naturally occurring Aspergillus species, isolated from compost, solarised and non-solarised soils, to control potato dry and pink rots caused respectively by F. sambucinum and P. erythroseptica, was evaluated in vitro and in vivo.
Tested using dual culture method, all the nine isolates of Aspergillus spp. tested, had inhibited the mycelial growth of F. sambucinum and P. erythroseptica by 27 to 68% and 16 to 25%, respectively. These findings confirm previous studies showing that native compost and soil-borne Aspergillus spp. significantly reduced the in vitro growth of Pythium spp., causing potato leak as well as that of many Fusarium species responsible for potato dry rot in Tunisia [11,29]. Furthermore, results revealed the inhibitory effects of Aspergillus species and isolates regardless of their origin of isolation. These findings are in accordance with several previous studies showing the potential antagonistic effects of Aspergillus isolated from many substrates. In fact, Barocio-Ceja et al.  showed that Aspergillus sp. “VC11” isolated from chicken-manure vermicompost inhibited the growth of F. oxysporum by 27%, that of F. subglutinans by 24% and that of Rhizoctonia sp. by 25%. Similarly, Israel and Lodha  reported the hyperparasitism of A. versicolor isolated from heated (naturally or solarized) cruciferous residue-amended soils against F. oxysporum cumini. Moreover, Adebola and Amadi  found that three Aspergillus species, namely A. niger, A. fumigatus et A. repens, isolated from the rhizosphere of black pod-infected cocoa trees showed ability to inhibit growth of Phytophthora palmivora in vitro and that the inhibition zones produced might be due to the production of antifungal metabolites by the test antagonists.
Our in vitro results showed that the highest inhibitory activity against both pathogens was induced by A. niger. These findings are in accordance with Dwivedi and Enespa  who found that among the biocontrol agents tested, A. niger isolated from tomato field was most effective and completely inhibited the mycelial growth of F. solani and F. oxysporum f. sp. lycopersici. In the same sense, Tiwari et al.  showed that A. niger inhibit the growth of all the fungal species tested which belong to ten genera of wood decay fungi (Trametes, Stereum, Pycnoporus, Phellinus, Lenzites, Phellinus, Earliella Gloeophyllum, Flavodon and Daedalea), from 29.2 to 66.7%. In addition, Venkatasubbaiah and Safeeulla  found that A. niger, isolated from the rhizosphere of coffee seedlings, was antagonistic to Rhizoctonia solani in vitro and exhibited hyperparasitism against this collar rot pathogen in dual culture experiments. The compost inhabiting A. niger was also able to inhibit the growth of soil-borne pathogens including Macrophomina phaseolina, Pythium aphanidermatum and Rhizoctonia solani . In addition, besides the inhibition of the mycelial growth, an atoxigenic A. niger FS10 isolated from Chinese fermented soybean was found to be able to inhibit spore germination and also to degrade the aflatoxin B1 of toxigenic A. flavus, and to reduce its mutagenicity and toxicity .
The suppressive effects of the microbial agents used in this study against potato pathogens may involve several mechanisms of action. In fact, to antagonize F. sambucinum and P. erythroseptica, Aspergillus isolates had probably deployed mycoparasitism, involving direct contact between the tested antagonist and the pathogens, (ii) production of antibiotic-type secondary metabolites, which spread through the medium, and (iii) competition for nutrients and space [63,64]. In fact, the first study showing that A. terreus can parasitize the sclerotia of Sclerotinia sclerotiorum under both laboratory and field conditions was reported by Melo et al. . Recently, the work of Hu et al.  clearly demonstrates that the mycoparasite characteristics of Aspergillus sp. ASP- 4 may be enhanced and related to its ability to produce a range of extracellular enzymes, such as chitinases and other antifungal extrolites which help colonization of S. sclerotiorum sclerotia; as well as thought to be the mode of action of A. terreus (Aspergillus section Terrei). In addition, chitinases have been shown to be produced by several Aspergillus spp. . In fact, antifungal activity of crude and purified chitinase produced by A. niger LOCK 62 was observed against F. culmorum, F. solani, and R. solani. The growth inhibition of F. culmorum was the strongest both by crude and purified enzymes (70 and 60%, respectively) whereas the growth of F. solani was strongly inhibited by crude chitinase (73%) . Recently, Frisvad and Larsen  mentioned that Aspergillus species are usually very efficient specialized metabolite producers which produces a wide array of small molecule extrolites (secondary metabolites or specialized metabolites), but also other bioactive molecules. Zhang et al.  have also isolated an endophytic A. clavatonanicus from a twig of Chinese Taxus mairei able to produce clavatol and patulin which exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, F. oxysporum f. sp. cucumerinum, R. solani, and P. ultimum. Recently, Patil et al.  have identified an isolate of A. flavus, as an endophytic fungus from Indian medicinal plant Aegle marmelos, able to produce phenolic and bioactive metabolite, characterized to be a flavonoid rutin with excellent biological activities.
Tested using the inverse double culture method, Aspergillus spp. isolates exhibited antifungal activity toward F. sambucinum and P. erythroseptica. The reduction of mycelial growth of both pathogens by Aspergillus spp. isolates involved the presence of volatile metabolites produced by each active antagonist tested. In the same sens, Gao et al.  revealed the presence of nine volatile metabolites produced by Aspergillus spp. (A. fumigatus, A. versicolor, A. sydowi, A. flavus, A. niger). In addition, Israel and Lodha  reported that, apart from releasing antibiotic substances, A. versicolor also produces volatile metabolites such as various hydrocarbons, alcohols, ketones, ethers, esters and sulphur-containing compounds. Moreover, volatile metabolites, emitted by A. niger, had been shown to inhibit the growth of Colletotrichum gloeosporioides . Recently, Thakur and Harsh  demonstrates that A. niger, isolated from the phylloplane of healthy Piper longum plants, possess higher antagonistic efficacy in inhibiting the mycelial growth of C. gloeosporioides in vitro by producing volatile metabolites.
Our results make clear that the inhibitory effect of Aspergillus spp. against F. sambucinum and P. erythroseptica in vitro varied depending on species and isolates of the same antagonist. In fact, populations of A. flavus in agricultural fields consist of complex communities that exhibit considerable genetic diversity based on phylogenetic and vegetative compatibility group analyses [70,71]. Natural communities of A. flavus consist of individuals that vary widely in ability to produce aflatoxin . In the same sense, Khan and Anwer  mentioned that the effectiveness of soil aggregate isolates of A. niger against R. solani varies with the isolate. In fact, the HCN-producing isolates AnC2 and AnR3, belonging to group I, produced greater amounts of siderophore and solubilized phosphorus than the other isolates and caused the greatest inhibition of colonization by R. solani in dual culture. They also suppressed the root rot on eggplant and the soil population of R. solani in pot soil .
The dry and pink rots are serious post-harvest potato diseases that cause important storage losses in Tunisia [7,9]. In the present study, four species of Aspergillus were used as antagonistic agents against the causative agents of these two tuber diseases. Results indicate that all the Aspergillus spp. tested significantly reduced the lesion diameter of the dry and pink rots as well as the average penetration of F. sambucinum and P. erythroseptica, whatever the timing of their application. In fact, the antagonistic potential of Aspergillus sp. against both diseases confirmed previous local reports on biocontrol of soil-borne pathogens with Aspergillus sp. isolated from compost teas [11,28-29]. Furthermore, efficacy of Aspergillus species in controlling fungal diseases has been proved. In fact, Aspergillus niger has a fair capacity to suppress plant pathogens and to increase the yield of the plants it colonizes [74,73]. In this sens, Khan and Anwer  reported that soil application of A. niger aggregate isolates, especially AnC2 and AnR3, suppressed R. solani infection and also significantly increased eggplant yield. The production of NH3, HCN and siderophore may have contributed to the suppression of R. solani. Hu et al.  mentioned also that their results strongly suggest that Aspergillus sp. ASP-4 has huge potential to be a successful biocontrol agent for Sclerotinia stem rot of oilseed rape. In fact, this antagonist significantly inhibits S. sclerotiorum by parasitism of the sclerotia, resulting in a marked decrease in the formation of apothecia in the field. In addition, Dwivedi and Enespa  reported that Aspergillus species were the most effective antagonists followed by Penicillium spp. and Trichoderma spp. for controlling the wilt of tomato and brinjal crops. In addition, the use of these bio-agents are not only safe for the farmers and consumers, but also eco-friendly, cost effective, easy to produce and easy to apply the formulations . Furthermore, results obtained by Wang et al.  indicated that the ASD strain, A. flavipes, could be useful as a potential biocontrol agent against the soil-borne fungus Phytophthora capsici and suggest that the increased activity of defence-related enzymes might be part of the mechanism of A. flavipes in controlling P. capsici. In other studies, Chuang et al.  reported that, based on pot experiments, significant increases in plant (Brassica chinensis Linn.) dry weight and N and P contents were observed with the addition of isolate 6A of A. niger, which has the ability to solubilise the native soil phosphate, which is not available to plant.
The present study also highlighted the importance of the timing of antagonist’s application on their effectiveness in dry and pink rots control. It was clearly evidenced that these compost and soil-borne fungi are able to significantly reduce rot development and severity when applied preventively following tuber wounding. In fact, when applied 24 h before inoculation with the pathogen, the Aspergillus isolates tested reduced more effectively the severity of Fusarium dry rot and pink rot. Similarly, Aydi et al.  recorded a better antagonistic effect of Aspergillus spp. when applied 24 h before inoculation with Pythium ultimum on potato tubers. Similar findings were reported by Daami-Remadi et al.  who used Aspergillus spp., Penicillium sp. and Trichoderma sp. as antagonistic agents against P. ultimum and P. aphanidermatum. Aydi-Ben Abdallah et al.  demonstrated that an important decrease in pink rot severity was achieved using preventive treatments of potato tubers with the culture filtrates and organic extracts from Aspergillus spp. In the same sense, applied preventively (24, 48 and 72 h before inoculation with the pathogen), dry rot severity incited by F. sambucinum was also limited using Bacillus spp. isolates  suggesting that this factor (i.e. treatment timing) may have a direct effect on disease control efficiency. In fact, Mohale et al.  reported that the timing of application of atoxigenic Aspergillus strains relative to the onset of host tissue colonisation by toxigenic strains, the innate competitive ability of the atoxigenic strains in the presence of their toxigenic counterparts are all important for the successful implementation of control strategies.
Aspergillus spp., isolated from soil and compost, exhibited an interesting antifungal activity against F. sambucinum and P. erythroseptica in vitro, using the dual culture and the inverse double culture methods, showing that these antagonists are able to release volatile and non-volatile metabolites. Thus, these Aspergillus species may represent a potential source of biologically active compounds and constitute a promising alternative to manage these increasingly important diseases.
Our studies demonstrated that Aspergillus species are potential native biological control agents against both potato diseases. They have shown great promise in their ability to reduce potato dry and pink rots. Their effectiveness was proved under extremely favourable conditions for F. sambucinum and P. erythroseptica development, such as use of susceptible potato cultivar (cv. Spunta), tuber wounding before inoculation, incubation under high moisture and disease conducive temperature, altogether have normally favoured pathogen expression and consequently, rot development.
Our results suggest that Aspergillus spp. are successful biocontrol agents for potato tuber rots in storage. These Aspergillus spp. may be further stimulated or enhanced by soil amendments and fertilizers, which result in the release of volatile chemicals that are toxic to potato pathogens (e.g., ammonia and glucosinolates), but can stimulate the growth and proliferation of Aspergillus species and other soil-borne mycoparasites [78,79].
This work was funded by the Ministry of Higher Education and Scientific Research of Tunisia through the funding allocated to the research unit UR13AGR09-Integrated Horticultural Production in the Tunisian Centre-East. My sincere gratitude goes to all the staff of the Regional Centre of Research in Horticulture and Organic Agriculture (CRRHAB) for their welcome and pleasant working conditions.