In Vitro Study on Influence of Some Streptomyces Strains Isolated from Date Palm Rhizosphere Soil on Some Toxigenic Fungi

Abstract

Date palm (Phoenix dactylifera L.) is considered the main crop in deserts and arid areas such as Saudi Arabia. Five Streptomyces species and other fungal species were isolated from date palm rhizosphere soil of several cultivars, such as Barhi, Khalas, Sullaj, and Sukkari, in the Ghat and El-Gouf regions. Streptomyces strains were isolated on Biolog universal growth agar medium and were identified following Biolog methods. The predominant Streptomyces isolated from this present survey was S. plicatus followed by S. rimosus, S. rutgersensus, and S. griseus. The number of soilborne fungi in the tested soil decreased with the increased number of isolated Streptomyces. There was a significant positive correlation between the date palm cultivar and the number of isolated Streptomyces. The in vitro effects of isolated Streptomyces species on some toxigenic fungi were also studied. Enzyme-linked immunosorbent assay, immunoaffinity column chromatography, and high-performance liquid chromatography were used to study the mycotoxins. The concentration of most of the tested mycotoxins was reduced significantly with the presence of the Streptomyces isolates. Results indicate that some Streptomyces isolates established in date palm rhizosphere have the ability to reduce production of mycotoxins of some toxigenic fungi; thus they have the potential of reducing the subsequent disease occurrence. Therefore they can be applied in wider range as biocontrol agents.

Introduction

D ate palm (Phoenix dactylifera L.) is one of the staple crops in desert areas where it is of high nutritional and economic values. It is also one of the oldest cultivated crops in arid areas with harsh environmental conditions. The number of date palms is about 100 million worldwide, of which 62 million can be found in the Arab world. Date palm is a palm extensively cultivated for its edible fruits. Because of its long history of cultivation, its exact native distribution is unknown and most likely originated somewhere in the desert oases of northern Africa, and perhaps also southwest Asia (Al Jasser, 2010). On a commercial scale, the Middle East and North Africa are the major date palm-producing areas in the world. Normally, palm is cultivated for subsistence/local markets on small farms alongside other crops because of its nutrition, great yields, and its long life (possibly up to 100 years). Date palm fruits are rich in carbohydrates, amino acids, fatty acids, salts, vitamins, minerals, and dietary fiber. World production of date palm fruits increased by 2.9% during the past 40 years (Barreveld, 1993; Al-Shahib and Marshall, 2003; Erskine et al., 2004).

The Kingdom of Saudi Arabia has an estimated 25 million date palms producing nearly a million tons of dates annually, which represents about 15% of the global date production. Date palms cover approximately 72% of the total area under permanent cropping. More than 400 different date palm cultivars are reported to exist in Saudi Arabia (Anonymous, 2006, 2009; Al-Abbad et al., 2011).

Roots of date palm trees may be exposed to attacks by many pathogenic microorganisms, whether bacteria, fungi, or Actinomyces. The rhizosphere area is rich in diverse and complex interactions between organisms. Several soilborne fungi that associated with date palm trees can cause root rot, wilt, and decline diseases. Date palm root rot may be caused by Fusarium solani and F. moniliforme, and necrotic date palm tops are caused by Botryodiplodia theobromae. Declined date palm roots are infected with F. oxysporum, F. solani, and F. moniliforme and also with Thielaviopsis paradoxa (Djerbi et al., 1986; Al-Roubaie et al., 1987; Abbas et al., 1989). The dominant fungi associated with date palm death and decline are F. oxysporum, Diplodia phoenicum, Ceratocystis radicicola, and Phomopsis phoenicola (Rattan and Al-Dboon, 1980; Mousiri et al., 2000).

Actinomyceyrs is considered one of the microorganisms presenting in high density in the rhizosphere. Previous studies indicated that 89% of the Actinomycetes isolates belonged to the genus Streptomyces. Only 11% were non-Streptomycetes, including Actinomadura sp., Microbispora sp., Micromonospora sp., Nocardia sp, Nonomurea sp., and three unclassified isolates. Twenty-three Streptomyces isolates showed activity against at least one of the five phytopathogenic fungi tested: Alternaria brassicicola, Collectotrichum gloeosporioides, F. oxysporum, Penicillium digitatum, and Sclerotium rolfsii (Khamna et al., 2009).

Since the discovery of mycotoxins in the 1960s, research has continued to find the most appropriate ways to reduce the risk of these toxins. There are many ways to reduce the risk of mycotoxins by detoxification-contaminated plant materials through various physical, chemical, and biological treatments. Many strains of Streptomyces, previously isolated from soil, demonstrated a strong and specific antagonistic effect against toxigenic fungi attributed to a proteinaceous compound (molecular size estimated to be 14 kDa) present in the supernatant of the culture. This compound showed nonchitinolytic fungicidal activity (Fulgueira et al., 2004).

This study aims to isolate some Streptomyces strains from date palm rhizosphere in different area of Saudi Arabia. Their impact on soilborne fungi species and enumeration and their effect on fungal ability to excrete different mycotoxins in vitro are also studied.

Materials and Methods

Sampling procedure

Date palm farming soil samples were obtained from different areas (Ghat and El-Gouf ) in Saudi Arabia at a depth of 20–25 cm. The samples were taken randomly over the year 2010 from the rhizosphere of many cultivars such as Barhi, Khalas, Sullaj, and Sukkari. Isolation, purification, and identification of microorganisms as well as mycotoxicology, detoxification trials, and statistical analysis were carried out in cooperation with the Plant Pathology Research Institute, Agricultural Research Center, Cairo, Egypt.

Isolation of Streptomyces and fungi from farming soil samples

The samples were dried for 3 days at room temperature to reduce the bacterial flora with no harm to the growth of Streptomyces (Korn-Wendisch and Kutzner, 1992). One gram of air-dried soil was shaken in a flask containing 99 mL of distilled water, and serial dilutions were carried out and inoculated on starch casein solid medium (Kuster and Williams, 1964). The plates were incubated at 29±2°C until the sporulation of Streptomyces colonies occurred. The colonies (where the mycelia remained intact and the aerial mycelia and long spore chains were abundant) were picked up and transferred to starch nitrate medium (Lechevalier and Lechevalier, 1970). Fungal isolation was conducted with the same method on potato dextrose agar medium (soil not dried).

Identification of Streptomyces and fungal isolates

Pure cultures of Streptomyces were obtained from selected colonies for repeated subculturing. An agar disk of a grown Streptomyces culture was removed aseptically and placed on a Biolog universal growth medium plate. The incubation extended for 48 hours at 29°C and then identified by the Biolog system according to Smalla et al. (1998).

After further incubation at 30°C for 3 days, the fungi developed were purified using hyphal tips or the single-spore technique and then transferred to slant containing potato dextrose agar medium. The purified fungi were verified and identified according to the procedures of Barnett (1960), Subramanian (1971), and Tousson and Nelson (1976).

Determination of mycotoxins

The most frequent fungi in both Ghat and El-Gouf samples were chosen to determine their important mycotoxins. The selected fungal isolates were vaccinated with the tested Streptomyces (S. plicatus, S. rimosu, S. rutgersansus, and S. griseus) individually and in a mixed form on sucrose, magnesium sulfate, potassium nitrate, and yeast extract liquid medium for 7 days at 27±2°C. The control treatment was free from any Streptomyces isolates.

Aspergillus toxins

Five milliliters of fungal homogenized sample was mixed with 70% methanol (1:5 ratio) and was infiltrated through a glass filter paper. Five milliliters of the previously obtained suspension was diluted in a 1:3 ratio. A total of 20 mL of solution was added into the enzyme-linked immunosorbent assay columnn with an injector. An immunoaffinity column was used for the detection of aflatoxin. This column was composed of gel suspension covalently bound with monoclonal antibodies specific to aflatoxin B1, B2, G1, and G2. The standard preparation procedure for the aflatoxin control was also applied. Incubation duration was extended to 2 hours from 30 minutes. The Ridasoft Win program was used for data collection and measurement of aflatoxins according to the method previously described (Ozaslan et al., 2011).

Fusarium toxins

Fumonisin toxins were determined according to the method described by Mazzani et al. (2001) as follows: Fifty grams of fungal ground sample of each isolate was blended with 5 g of sodium chloride and 100 mL of methanol:water (80:20 vol/vol) and were thoroughly mixed before filtration through glass microfiber filter papers. Ten milliliters of culture filtrate was diluted to 40 mL with wash buffer (0.1% Tween in phosphate-buffered saline) and then filtered through a microfiber filter (pore size, 1.0 μm). Ten milliliters of this diluted filtrate was passed through a fumontest column (Vicam Co., USA). The fumonisin was eluted from column with 1 mL of methanol. One milliliter of developer A (No. G5005) and developer B (No. G5004) was added to the methanol elute, and fluorescence was then measured with a fluorometer (Series-4). Zearalenone and T-2 toxins were quantified by the same protocol mentioned by Martins et al. (2003) except the separation column type and dilution rate were adjusted with 49 mL of distilled water.

Alternaria toxins

Fifty milliliters of crude extract was transferred to a blender cup with the help of 150 mL of methanol. It was blended with gentle irritation for 3 minutes and transferred to a glass funnel fitted with a fluted filter paper. An additional 50 mL of methanol was used for washing the residues left in the blender cup into the filter paper. An aliquot of 200 mL of the filtrate was collected into a beaker, and 60 mL of 10% ammonium sulfate solution was added. The mixture was filtered through fluted filter paper. An aliquot of 200 mL or less of the filtrate was transferred to a separating funnel, and 50 mL of water at 8°C or below was added. Two extractions with 40 mL of chloroform, shaking for 2 minutes each time, were conducted. All the chloroform was collected in a separating funnel and washed with 30 mL of ultrapure water at 5–8°C. The chloroform was then transferred to a graduated cylinder, and the volume was measured for future calculations. The chloroform extract was evaporated in a rotary evaporator at 35°C. The residue was dissolved in 2 mL of methanol and filtered through anhydrous sodium sulfate. The high-performance liquid chromatography system consisted of a model HP 1050 liquid chromatograph. The analytical column was Spherisorb ODS-2 (particle size, 5 mm; length, 250 mm). The mobile phase was methanol/water (80:20 vol/vol) containing 300 mg of ZnSO₄·H₂O/L, while the flow ratio was 0.7 mL/minute. The wavelength for recording chromatograms was 250 nm. A calibration curve was constructed for quantification purposes using the toxin standards according to Da Motta and Valente Soares (2000).

Mycotoxin detoxification in vitro

Under the same condition, the selected isolates (Alternaria alternata, Aspergillus parasiticus, F. solani, and F. oxysporum) were grown on sucrose, magnesium sulfate, potassium nitrate, and yeast extract liquid medium with S. plicatus, S. rimosu, S. rutgersensus, and S. griseus individually and also as a mixture. Each treatment was incubated at 27±2°C for 7 days in three replicates. Then different mycotoxins were determined as mentioned above.

Statistical analysis

The data obtained were statistically analyzed using ANOVA with the MSTAT-C statistical package. The least significant difference procedure was used at the 0.05 level of probability (Fisher, 1948).

Results

The results (Table 1) showed that 141 fungal isolates belonging to 14 genera and 28 species were isolated from Ghat and El-Gouf soil samples. Seventy-five fungal species were isolated from the rhizosphere soil of date palm cultivars of Barhi, Khalas, Sullaj, and Sukkari in Ghat. As. parasiticus and F. solani were the highest occurring species at 8.0% each, whereas Alternaria sp., Chaetosphaeropsis sp., F. equiseti, Mycosphaerella sp., P. chrysogenum, and Phoma sp. were the lowest at 1.3%. The rhizosphere soil of the Sullaj cultivar had more fungal isolates (31) than the other cultivars; Barhi, Khalas, and Sukkari had 19, 10, and 15 isolates, respectively. In El-Gouf, 66 fungal isolates were collected. The most frequent were Al. alternata and F. oxysporum (9.1%), whereas the lowest frequency was seen for Chaetomium atrobrunneum, Phoma spp., and Gliocladium spp. (15%). The rhizosphere soil of the Khalas cultivar was less infectious, followed by Sukkari, Barhi, and Sullaj.

Table 1.

Occurrence and Frequency of Fungi Isolated from Rhizosphere Soil of Different Date Palm Cultivars on Potato Dextrose Agar Medium After 7 Days of Incubation at 27±2°C

	Ghat						El-Gouf
	Barhi	Khalas	Sullaj	Sukkari	No. of isolates	% of frequency	Barhi	Khalas	Sullaj	Sukkari	No. of isolates	% of frequency
Fungal isolate
Al. alternata	1	1	1	1	4	5.3	2	1	2	1	6	9.1
Alternaria sp.	0	0	1	0	1	1.3	1	0	1	0	2	3.0
As. flavus	1	0	1	1	3	4.0	1	0	2	1	4	6.1
As. niger	1	1	2	1	5	6.7	1	0	1	1	3	4.5
As. fumigatus	2	0	1	0	3	4.0	0	1	2	0	3	4.5
As. parasiticus	1	1	2	2	6	8.0	1	0	3	0	4	6.1
Aspergillus spp.	0	0	2	1	3	4.0	0	1	0	0	1	1.5
Botryodiplodia theobromae	1	0	1	0	2	2.7	1	0	2	1	4	6.1
Chaetomium atrobrunneum	1	0	1	0	2	2.7	0	0	1	0	1	1.5
Chaetosphaeropsis spp.	0	1	0	0	1	1.3	0	0	0	0	0	0.0
Diplodia spp.	1	0	1	1	3	4.0	1	0	1	0	2	3.0
Fusarium sp.	1	0	2	1	4	5.3	0	1	1	0	2	3.0
F. equiseti	1	0	0	0	1	1.3	1	0	1	1	3	4.5
F. moniliforme	1	2	0	1	4	5.3	1	0	1	0	2	3.0
F. semitectum	0	0	2	1	3	4.0	1	1	1	0	3	4.5
F. solani	1	1	2	2	6	8.0	1	0	0	1	2	3.0
F. oxysporum	1	0	3	1	5	6.7	2	1	2	1	6	9.1
Gliocladium spp.	0	0	0	0	0	0.0	0	0	1	0	1	1.5
Graphiola phoenicis	0	0	0	0	0	0.0	0	0	0	0	0	0.0
Mycosphaerella spp.	0	0	1	0	1	1.3	0	0	0	0	0	0.0
Pencillium sp.	2	0	2	0	4	5.3	0	1	1	0	2	3.0
P. notatum	0	1	0	0	1	1.3	1	0	1	1	3	4.5
P. italicum	0	1	1	1	3	4.0	0	0	2	0	2	3.0
P. chrysogenum	0	0	1	0	1	1.3	1	0	0	1	2	3.0
P. expansum	1	0	3	1	5	6.7	0	0	2	0	2	3.0
Phoma sp.	0	0	1	0	1	1.3	1	0	0	0	1	1.5
Phomopsis sp.	1	0	0	0	1	1.3	0	0	1	1	2	3.0
Thielaviopsis paradoxa	1	1	0	0	2	2.7	1	0	1	1	3	4.5
Total	19	10	31	15	75		18	7	30	11	66

Ghat had 14 Streptomyces strains isolated (Table 2). S. plicatus was the most frequent at 35.7%, followed by S. rimosu at 21.4%, whereas the lowest frequency was 7.1% for both S. rutgersensus and S. griseus. However, the frequency of Streptomyces spp. was 28.6%. The highest number of Streptomyces isolates was gained from the soil of Khalas cultivars, whereas the lowest numbers were found from the soil of the Sullaj cultivar. There were 18 strains of all the Streptomyces isolated from the El-Gouf area, of which seven strains were S. plicatus, four strains were S. rimosu, two strains were S. rutgersensus, one strain was S. griseus, and four strains were Streptomyces spp. Most strains of Streptomyces were concentrated in the Khalas rhizosphere, followed by the Sukkari, Barhi, and Sullaj rhizospheres with eight, five, three, and two, respectively.

Table 2.

Occurrence and Frequency of Streptomyces Strains from Rhizosphere Soil of Different Date Palm Cultivars on Biolog Universal Growth Agar Medium After 2 Days of Incubation at 29±2°C

	Ghat						El-Gouf
Streptomyces spp.	Barhi	Khalas	Sullaj	Sukkari	No. of isolates	% of frequency	Barhi	Khalas	Sullaj	Sukkari	No. of isolates	% of frequency
S. plicatus	1	3	0	1	5	35.7	1	3	1	2	7	38.9
S. rimosu	1	1	0	1	3	21.4	1	2	0	1	4	22.2
S. rutgersensus	0	1	0	0	1	7.1	0	1	0	1	2	11.1
S. griseus	0	1	0	0	1	7.1	0	1	0	0	1	5.6
Streptomyces sp.	1	1	1	1	4	28.6	1	1	1	1	4	22.2
Total	3	7	1	3	14		3	8	2	5	18

Data in Table 3 show isolate number 6 of As. parasiticus excreted more total aflatoxins than the other isolates (22 ppb), whereas numbers 1, 4, and 5 were equal in aflatoxin B1 (11 ppb). S. plicatus was the most effective in aflatoxin detoxification, especially for As. parasiticus number 4 and followed by S. rimosu and S. rutgersensus, whereas S. griseus was the least effective with the tested As. parasiticus isolates except number 5.

Table 3.

Amount of Total Aflatoxins and Aflatoxin B1 Produced by Six Isolates of Aspergillus parasiticus Obtained from Ghat, Grown on Sucrose, Magnesium Sulfate, Potassium Nitrate, and Yeast Extract Liquid Medium for 7 Days at 27±2°C, and Detoxification Effect of Some Different Streptomyces Strains

	Total aflatoxins and B1 toxins (ppb) excreted by As. parasiticus and detoxification of different Streptomyces strains
As. parasiticus isolate, toxin measurement	Control	S. plicatus	S. rimosu	S. rutgersensus	S. griseus	Mixture
No. 1
B1	11	4	6	5	5	3
Total	20	7	10	11	13	6
% of reduction	0	65.0	50.0	45.0	35.0	70.0
No. 2
B1	10	3	5	7	6	3
Total	21	8	9	12	14	9
% of reduction	0	61.9	57.1	42.9	33.3	57.1
No. 3
B1	10	5	7	7	8	6
Total	19	7	9	8	10	7
% of reduction	0	63.2	52.6	57.9	47.4	63.2
No. 4
B1	11	2	6	6	7	3
Total	20	6	9	11	12	6
% of reduction	0	70.0	55.0	45.0	40.0	70.0
No. 5
B1	11	4	5	5	4	3
Total	19	8	10	12	11	7
% of reduction	0	57.9	47.4	36.8	42.1	63.2
No. 6
B1	10	3	3	6	8	4
Total	22	7	9	10	12	7
% of reduction	0	68.2	59.1	54.5	45.5	68.2

The percentage of reduction with the least significant difference test at 5% was 3.34%.

F. solani number 2 produced more Fusarium toxins (fumonisin, zearalenone, and T-2 toxins) than other isolates. The most effective detoxification occurred with S. plicatus, whereas the least effective was S. griseus with F. solani isolates numbers 6 and 4, respectively (Table 4).

Table 4.

Amount of Fumonisin, Zearalenone, and T-2 Toxin Produced by Six Isolates of Fusarium solani Obtained from Ghat, Grown on Sucrose, Magnesium Sulfate, Potassium Nitrate, and Yeast Extract Liquid Medium for 7 Days at 27±2°C, and Detoxification Effect of Some Different Streptomyces Strains

	Fumonisin, Zearalenone, and T-2 toxin (ppb) excreted by F. solani and detoxification of different Streptomyces strains
F. solani isolate, toxin measurement	Control	S. plicatus	S. rimosu	S. rutgersensus	S. griseus	Mixture
No. 1
Fumonisin	1050	260	280	355	325	255
Zearalenone	150	85	100	120	135	83
T-2	50	20	27	35	30	19
Total	1250	365	407	510	490	357
% of reduction	0.0	70.8	67.4	59.2	60.8	71.4
No. 2
Fumonisin	1200	340	390	425	440	320
Zearalenone	325	115	135	200	185	110
T-2	70	25	35	40	38	23
Total	1595	480	560	665	663	453
% of reduction	0.0	69.9	64.9	58.3	58.4	71.6
No. 3
Fumonisin	950	285	310	315	325	283
Zearalenone	190	75	89	110	98	72
T-2	90	42	49	57	60	40
Total	1230	402	448	482	483	395
% of reduction	0.0	67.3	63.6	60.8	60.7	67.9
No. 4
Fumonisin	1300	410	460	485	530	408
Zearalenone	220	70	69	84	90	66
T-2	40	14	18	24	21	14
Total	1560	494	547	593	641	488
% of reduction	0.0	68.3	64.9	62.0	58.9	68.7
No. 5
Fumonisin	650	205	215	227	238	200
Zearalenone	250	88	89	125	120	86
T-2	50	18	21	29	27	14
Total	950	311	325	381	385	300
% of reduction	0.0	67.3	65.8	59.9	59.5	68.4
No. 6
Fumonisin	1100	198	212	240	261	195
Zearalenone	240	55	61	66	78	55
T-2	30	11	18	19	19	11
Total	1370	264	291	325	358	261
% of reduction	0.0	80.7	78.8	76.3	73.9	80.9

The percentage of reduction with the least significant difference test at 5% was 1.64%.

Table 5 and Figure 1 show that Al. alternata isolate number 3 produced the highest concentration of alternariol (AOH) toxins (15 ppb). Both isolates numbers 3 and 6 excreted the highest concentration of AOH monomethyl ether (AME) toxin (9 ppb) compared with the other tested isolates. However, Alternaria toxins decreased following inoculation with the tested Streptomyces strains, especially S. plicatus. The total toxin reduction reached to 69.6% with Al. alternata number 1. The detoxification ratio decreased to 22.1% with Al. alternata isolate number 4 when inoculated with S. griseus.

FIG. 1.

High-performance liquid chromatography analysis of alternariol (AOH) and alternariol monomethyl ether (AME) produced by different isolates of Alternaria alternata grown on sucrose, magnesium sulfate, potassium nitrate, and yeast extract liquid medium and treated with different strains of Streptomyces for 7 days at 27±2°C to detoxify these mycotoxins. AU, absorbance units.

Table 5.

Amount of Alternariol and Alternariol Monomethyl Ether Produced by Six Isolates of Alternaria alternata Obtained from El-Gouf, Grown on Sucrose, Magnesium Sulfate, Potassium Nitrate, and Yeast Extract Liquid Medium for 7 Days at 27±2°C, and Detoxification Effect of Some Different Streptomyces Strains

	AOH and AME (ppb) excreted by Al. alternata and detoxification of different Streptomyces strains
Al. alternata isolate, toxin measurement	Control	S. plicatus	S. rimosu	S. rutgersensus	S. griseus	Mixture
No. 1
AOH	14.5	4	4.5	5.8	5.6	3.5
AME	8.5	3	3.5	4.2	4	2.5
Total toxins	23	7	8	10	9.6	6
% of reduction	0.0	69.6	65.2	56.5	58.3	73.9
No. 2
AOH	10.5	3.5	4.1	6.2	6	3.5
AME	6.5	2.5	3	3.7	3.5	1.9
Total toxins	17	6	7.1	9.9	9.5	5.4
% of reduction	0.0	64.7	58.2	41.8	44.1	68.2
No. 3
AOH	15	6.5	6.5	8.4	8.8	4.5
AME	9	5.5	6	6.9	6.7	3.5
Total toxins	24	12	12.5	15.3	15.5	8
% of reduction	0.0	50.0	47.9	36.3	35.4	66.7
No. 4
AOH	8	5	6.8	7.8	7.6	4
AME	6	2	2.6	3.5	3.3	1.6
Total toxins	14	7	9.4	11.3	10.9	5.6
% of reduction	0.0	50.0	32.9	19.3	22.1	60.0
No. 5
AOH	11	5	5.9	6.9	7.1	3.8
AME	7	3	3.6	4.2	3.8	2.8
Total toxins	18	8	9.5	11.1	10.9	6.6
% of reduction	0.0	55.6	47.2	38.3	39.4	63.3
No. 6
AOH	12	6.5	7.8	8.4	7.9	4.1
AME	9	3.5	4.2	5.6	5.5	3
Total toxins	21	10	12	14	13.4	7.1
% of reduction	0.0	52.4	42.9	33.3	36.2	66.2

The percentage of reduction by the least significant difference test at 5% was 1.97%.

AOH, alternariol; AME, alternariol monomethyl ether.

F. oxysporum isolate number 3 excreted the highest concentration of total Fusarium toxin and zearalenone (1709 and 353 ppb, respectively) (Table 6). However, the highest concentration of fumonisin and T-2 toxin was produced by F. oxysporum number 5 and F. oxysporum number 4 at 1413 and 98 ppb, respectively. The highest reduction of total toxins was 70.5%, reached by S. plicatus with F. oxysporum isolate number 2. In contrast, the lowest reduction occurred for S. griseus when inoculated with tested isolate F. oxysporum number 1.

Table 6.

Amount of Fumonisin, Zearalenone, and T-2 Toxin Produced by Six Isolates of Fusarium oxysporum Obtained from El-Ghouf, Grown on Sucrose, Magnesium Sulfate, Potassium Nitrate, and Yeast Extract Liquid Medium for 7 Days at 27±2°C, and Detoxification Effect of Some Different Streptomyces Strains

	Fumonisin, zearalenone, and T-2 toxin (ppb) excreted by F. oxysporum and detoxification of different Streptomyces strains
F. oxysporum isolate, toxin measurement	Control	S. plicatus	S. rimosu	S. rutgersensus	S. griseus	Mixture
No. 1
Fumonisin	815	253	290	333	441	239
Zearalenone	217	99	118	178	161	100
T-2	43	21	25	30	27	21
Total	1076	373	433	541	629	360
% of reduction	0.0	65.4	59.7	49.7	41.5	66.6
No. 2
Fumonisin	1141	286	301	394	349	277
Zearalenone	163	93	108	133	145	90
T-2	54	22	29	39	32	21
Total	1359	401	438	567	527	388
% of reduction	0.0	70.5	67.8	58.3	61.2	71.4
No. 3
Fumonisin	1280	374	419	472	473	348
Zearalenone	353	126	145	222	199	120
T-2	76	27	38	44	41	25
Total	1709	527	602	739	713	492
% of reduction	0.0	69.1	64.8	56.8	58.3	71.2
No. 4
Fumonisin	1033	313	333	350	349	308
Zearalenone	207	82	96	122	105	78
T-2	98	46	53	63	65	43
Total	1337	442	482	536	519	429
% of reduction	0.0	67.0	64.0	59.9	61.2	67.9
No. 5
Fumonisin	1413	451	495	539	570	443
Zearalenone	239	77	74	93	97	72
T-2	43	15	19	27	23	15
Total	1696	543	588	659	689	530
% of reduction	0.0	68.0	65.3	61.2	59.4	68.7
No. 6
Fumonisin	707	225	231	252	256	217
Zearalenone	272	97	96	139	129	93
T-2	54	20	23	32	29	15
Total	1033	342	349	423	414	326
% of reduction	0.0	66.9	66.2	59.0	59.9	68.4

The percentage of reduction with the least significant difference test at 5% was 2.25%.

Discussion

The current study demonstrated differences in species and number of fungi isolated from Ghat and El-Gouf rhizosphere soil. This might be due to differences in the characteristics of soil, pH, fertilization, humidity percentage, root secretions, microbial competition, and climatic conditions as well as date palm cultivars, tree age, and plant density per unit area. These variations were in agreement with the findings of Albiach et al. (2000), Broeckling et al. (2008), and Ndubuisi-Nnaji et al. (2011). The most common fungal isolates were As. parasiticus, F. solani, As. flavus, F. oxysporum, P. expansum, As. niger, and B. theobromae at both locations of Ghat and El-Gouf. These results were consistent with those reported by Mousiri et al. (2000) and Abdullahi et al. (2010).

Isolation for fungi and actinomyces from soil samples of Khalas, Sukkari, Barhi, and Sullaj cultivars was carried out under the same conditions; however, different media and incubation conditions were adopted for different cultivars. A negative association was found between the number and the species of isolated fungi and Streptomyces in the areas of both Ghat and El-Gouf. These results might be due to the fact of antibiosis in some Streptomyces against a wide range of fungi in date palm rhizosphere. These findings were in agreement with some previous results (Haskell et al., 1958; Lockwood, 1964; Errakhi et al., 2007). The most common Streptomyces strains in both regions were S. plicatus, S. rimosu, S. rutgersensus, and S. griseus, and these findings were in agreement with some previous studies (Carvajal, 1946; Abd-Allah, 2001; Hozzein et al., 2011).

All the tested Streptomyces strains showed the ability to reduce the total aflatoxin and B1 produced by the tested As. parasiticus isolates. However, differences were found in the reduction of different strains, which might be explained as relative physiological and genetic differences between isolates. These findings were agreed with a previous report (Hassan et al., 2011). The same detoxification trend of these tested Streptomyces extended to both F. oxysporum and F. solani toxins (fumonisin, zearalenone, and T-2 toxins) as well as Al. alternata toxins (AOH and AME). All these findings were similar to previous reports (Karlovsky, 1999; Alkahtani Muneera et al., 2011).

In vitro the tested Streptomyces strains had a clear role to reduce levels of the aflatoxin B1, total aflatoxin, fumonisin, zearalenone, T-2 toxin, AOH, and AME produced by all tested fungi. The S. plicatus strain was more efficient than the other strains. These findings were in harmony with previous reports (Sakuda et al., 1996; Zucchi et al., 2008). This efficiency to detoxify the mycotoxins may be due to many possible reasons. For example, the Streptomyces may have a genetic structure that can work in certain enzyme systems, working on breaking down these mycotoxins, through secreted chemical substances that interact with these toxins and form new compounds needed for Streptomyces cell formation and metabolism or antagonistic behavior of these Streptomyces strains.

Conclusions

This results revealed the fungal diversity in the soil surrounding the roots of some date palm cultivars. Khalas and Sukkari varieties were less frequently affected by fungi, followed by Barhi and Sullaj. Therefore, to avoid some fungal infection, planting Khalas and Sukkari cultivars is recommended. In addition, there were some strains of Streptomyces that were associated with some types of palm cultivars. Laboratory studies showed the ability of Streptomyces to reduce the amount of mycotoxins produced as aflatoxins, fumonisin, zearalenone, T-2, AOH, and AME toxins. There was variation in the ability of these Streptomyces strains to detoxify: S. plicatus isolates were the most efficient, and S. griseus isolates were the weakest. The active strains can be used as bioagents to detoxify mycotoxins in animal and poultry feed and/or can be used on different crops to reduce the mycotoxin contamination ratio, although this trend needs further study.

Footnotes

Acknowledgments

The authors wish to thank Dr. Eman M. Abdelkareem (Plant Pathology Research Institute, Egypt), Dr. Allahdino Channa (General Organization for Grain Silos and Flour Mills, Saudi Arabia), Prof. Dr. Mohamed Reda El-Tohamy (Zagazig University, Egypt), and Dr. Guy Mozolowski (University of Nottingham, United Kingdom) for revision of this manuscript.

Disclosure Statement

No competing financial interests exist.

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