Management of date palm root rot diseases by using some biological control agents under organic farming system

In the current work an attempt was made to find out the most suitable bioagents that have the ability to protect Date palm cv. Zaghloul ( Phoenix dactylfera L.) from some soilborne fungal diseases. Several soilborne fungi were isolated from root rots of date palm trees located in the farms of El-Beheira Governorate, Egypt, including; Fusarium solani , F. oxysporum , Rhizoctonia solani and Macrophomina phaseolina , and their pathogenicity were confirmed on date palm seedlings in the greenhouse. These fungi cause economic losses in date palm yield and a wide range of other cultivated plants. Many different antagonistic isolates (bioagents) i.e. Trichoderma album , T. harzianum , T. viride and T. hamatum were isolated from the rhizosphere soil of healthy date palm trees. For comparison of results, bio-commercial preparations mainly “Bio-zeid ( T. album 10×10 6 cfu/ ml)” and “Plant guard ( T. harzianum 30×10 6 cfu/ ml)” were also used to detect their antagonistic potential against the mycopathogens of date palm. In vitro antifungal efficacy of the bioagents was evaluated against all the pathogens, where T. harzianum was the most effective as it caused 87.10, 81.55, 77.60 and 68.55% reduction in the radial growth of F. solani , F. oxysporum , R. solani and M. phaseolina , respectively. In vivo assays under field conditions, all tested biotic treatments significantly reduced severity of root rot diseases caused by the concerned pathogens. Moreover, they increased the percentages of survived date palm plants in infested soils during both successive growing seasons of 2016 and 2017, where T. harzianum was the most effective bioagent as it showed an increase in date palm survival of about 82.35 and 86.67% at both seasons, respectively. In addition, all bioagents enhanced the growth parameters of date palm, i.e. plant height (cm), number of leaves/ plant and number of leaflets/ leaf, compared with the control treatment. Thus, these effective bioagents could be used as biofungicides to control the root rot diseases of date palm in the field; accordingly, we could displace the use of non-ecofriendly and health hazards synthetic fungicides.


Introduction
Date palm is one of the most valuable domesticated fruit trees because of its significance in human societies, health benefits, and the range of subsistence products from its fruits and other parts of the large palm (Johnson et al., 2015). According to Pariona, (2017), Egypt leads the world date palm production with about 1.1 million metric tons annual production and generated about $41.8 million from export of fresh date fruits.
Agricultural management of soilborne pathogens in the field include crop rotation, fungicides applications, methyl bromide fumigation, soil solarization and the use of resistant or tolerant varieties. However, no single method provided an adequate control of soilborne diseases (Hausbek and Lamour, 2004). Use of synthetic chemicals to control soilborne pathogens caused several negative effects on the plants such as: i) development of pathogen resistance, ii) harmful effects on humans, iii) bad impact on beneficial organisms, and iv) environmental pollution. However, soilborne pathogens still need to be controlled in order to ensure healthy plant growth and productivity in sustainable agriculture. Kaewchai and Soytong, (2010) stated that biological control of plant pathogens by using microorganisms has been considered more natural and environmentally acceptable alternative to the use of chemicals.
Trichoderma spp. are common saprophytic fungi which were found in almost all soils and among rhizosphere microflora. They have been used as potential biocontrol agents because of their ability to reduce the incidence of diseases caused by many soilborne pathogens (Abdel-Monaim, 2010;Perveen and Bokhari, 2012). Several strains of Trichoderma spp. have been isolated and were regarded as effective biocontrol agents against several phytopathogenic fungi under greenhouse and field conditions (Omomowoa et al., 2018).
Several reports on using nonpathogenic fungi as biocontrol agents were recorded such as; Chaetomium species for control Thielaviopsis bud rot of Hyophorbe lagenicaulis (Soytong et al., 2005), whereas, T. viride, T. polysporum, T. hamatum and T. aureoviride significantly reduced growth of Ceratocystis paradoxa (Eziashi et al., 2007). Modes of action of these beneficial microorganisms in suppressing plant pathogens include; direct parasitism of pathogens, competition for space and nutrients, the production of antibiotics, enzymes, and plant growth promoting hormones (Lugtenberg et al., 2003). Moreover, in the study of Abdel-Monaim, (2010); Perveen and Bokhari, (2012), these bioagents significantly increased root and plant growth of date palm and of many other crops.
The objectives of our work were; to decrease the use of chemical fungicides in agricultural fields of date palm to enhance the growth and yields of date palm. In addition, we made a trial to obtain the most promising bioagent that has the ability to protect date palm cv. Zaghloul plants from certain soilborne fungal pathogens.

Isolation and identification of the root rot pathogens of date palm trees
About 3-10 root pieces showing necrosis and/ or root rot symptoms were collected from naturally infected date palm trees (cv. Zaghloul) from the top 5 cm of the soil level. These trees were from different locations of EL-Beheira Governorate, Egypt during the growing season of 2016. According to Maciá-Novel Research in Microbiology Journal, 2018 Vicente et al., (2008), infected roots were washed, air dried, surface sterilized in 1% sodium hypochlorite solution for 3 min., washed several times with sterilized dist. water and then dried between two sterilized filter papers. The sterilized root fragments were aseptically transferred to the surface of plates of Potato dextrose agar medium (PDA). Plates were incubated at 25°C for 5 d and were examined daily. The developed mycelial growth was picked up and transferred onto new PDA medium. Purification of each isolated fungus was carried out using mycelium tip culture (Al-Sa'di et al., 2007).
Identification of the isolated fungi was carried out according to their cultural and microscopical characteristics described by Singh, (1982); Barnett and Hunter, (1987). Colony morphology, conidiophores and conidia of Fusarium were identified according to Nelson et al., (1983);Booth, (1985). Stock cultures were maintained on PDA slants and kept at 5°C till further studies.

Isolation and identification of the antagonistic mold fungi
Rhizosphere soil of healthy roots of date palm trees were used to isolate different antagonistic mold fungi using the method of Ahmed, (2005). Ten g of rhizosphere soil was added aseptically to 90 ml sterile dist. water (to make stock dilution of 10 -1 ), shacked on rotary shaker for 15 min. and then serially diluted up to 10 -4 . PDA medium supplied with 66.7 mg/ l rose bengal and 250 mg/ l streptomycin (Johnson et. al., 1960) was used for isolating the antagonistic fungi. One ml of each dilution was aseptically transferred to sterilized petri plates containing 20 ml of melted PDA medium, and then spread using sterile glass rod. Three plates were used for each dilution. All plates were incubated at 25±1ºC for 4 d. Separate colonies of the isolated fungi were selected, sub-cultured and identified according to their morphological and microscopical characteristics (Rifai, 1969).

Pathogenicity assay
This experiment was carried out in a greenhouse located in Environmental Studies and Research Institute, University of Sadat City, Minufiya Governorate, Egypt. Date palm seeds (cv. Zaghloul) were treated with dry heat at 45°C for 2h to activate their germination then planted in 30 cm diameter plastic pots (one seed per pot) filled with steam pasteurized soil. After 6 months of planting, seedlings were inoculated separately with each of the fungal pathogens using homogenized culture technique (Muthomi et al., 2007). Disks were taken from one-week-old cultures of the tested fungi and transferred to 75 ml of potato dextrose broth (PDB) in a 250 mL flask, and then incubated at 25 ± 1ºC for 6-7 d. The fungal mycelia were separated using sterile Whatman no. 1 filter paper, rinsed with sterile dist. water and then blended with a small amount of sterile water in a waring blender for 2 min. Sterile dist. water was then added to each fungal suspension to have a final concentration of 6×10 6 cfu/ ml for soil infestations. Ten pots were used for each fungal isolate along with non-infested negative control soil.
The severity of root rot symptoms was determined 90 d following pathogens inoculation using a disease rating scale of 0-5 on the basis of root discoloration and leaf yellowing as follows; 0: no root discoloration or leaf yellowing, 1: 1-25% root discoloration or one yellow leaf, 2: 26-50% root discoloration or more than one yellow leaf, 3: 51-75% root discoloration plus one wilted leaf, 4: up to 76% root discoloration or more than one wilted leaf, and 5: completely dead plants. For each replicate, a disease severity index (DSI) was calculated according to Abdullah et al., (2003)

In vitro antagonistic potency of the isolated mold fungi against root rot mycopathogens
The antagonistic activity of the different fungal isolates on the radial growth of the mycopathogens was conducted under laboratory conditions using poisoned food technique. T. album, T. hamatum, T. harzianum and T. viride isolated from rhizosphere soil of healthy date palm trees, and commercial preparations of "Bio-zeid (T. album 10×10 6 cfu/ ml)" and "Plant guard (T. harzianum 30×10 6 cfu/ ml)" were evaluated during this assay. Unless otherwise stated, the PDA medium was used for all the growing fungi. Culture filtrate of each of the fungal antagonists was prepared separately according to (Calistru et al., 1997). 1 ml of sterile cell free-filtrate of each of the antagonistic fungi and the commercial preparation suspensions were separately added to warm PDA and then poured into petri plates (10 ml/ plate). Each of these treated plates was inoculated separately at the center with discs cut from the periphery of 5 d old cultures of each of F. solani, F. oxysporum; R. solani, and M. phaseolina. Plates inoculated with pathogens only without antagonists served as positive control treatment, whereas, nontreated plates served as negative control. Inoculated plates were incubated at 25± 1°C for 7 d. Five plates were used for each treatment (Mishra, 2010).
The experiment was terminated when the mycelial mats covered the surface of PDA in negative control plates. All plates were observed, and percentage of reduction in the radial growth of the pathogenic fungi was calculated using the formula suggested by Ahmed, (2005); Ahmed, (2013) as follows: Where; G1: growth of the pathogenic fungus in positive control plates, G2: growth of pathogen in plates treated with fungal antagonists.

2.5.
Evaluation of efficacy of the bioagents to control root rot of date palm plants under field conditions

Preparation of inocula of the bioagents
In this assay, each of the antagonistic fungi was grown in PDB medium under dark conditions for 10 d at 25± 2ºC (Ahmed, 2013). All cultures were individually blended in an electrical blender for 2 min. then used as inocula at concentrations of 30×10 6 cfu/ ml.

In vivo assay
Field experiments were conducted in naturally infested farms with each of the fungal pathogens of root rot diseases separately, located in EL-Beheira Governorate, Egypt, during the two successive growing seasons of 2016 and 2017. These assays were carried out to evaluate the efficacy of the tested bioagents and the commercial preparations in controlling root rot diseases of date palm, as well as their effects on seedlings growth parameters.
The experimental design was a complete randomized block with 10 replicates. The experimental unit areas were 2m 2 (1×2 m), each unit included 2-year-old date palm plants. The soils of date palm were drenched 3 times at 15-d intervals with inocula of each of the bioagents (30×10 6 cfu/ ml) and the bio-commercial preparations "Bio-zeid, T. album 10×10 6 cfu/ ml", and "Plant guard (T. harzianum 30×10 6 cfu/ ml)" separately, at the rate of 3 l per offshoot. Soils infested with each of the 4 mycopathogens only separately, act as positive control treatments. Untreated negative control soils were drenched three times at 15-d intervals with water. The recommended agricultural practices and irrigation dates for date palm were used.
Novel Research in Microbiology Journal, 2018 The disease severity was assessed for each treatment after 6 months from the last application treatment as mentioned above according to Ilias, (2000). At the end of these field experiments the following plant growth parameters were estimated: Plant height (cm), number of leaves/ plant and leaflets numbers/ leaf.

Statistical analysis
Data were subjected to statistical analysis and compared according to the least significant difference (L.S.D.) as mentioned by Snedecor and Cochran, (1989).

Isolation, identification of the mycopathogens and rhizosphere mold fungi
Data in Table (1) indicated that, R. solani (31.58%) was the most frequently isolated fungus from the rotted samples of date palm trees. On the contrary, M. phaseolina (18.42%) recorded the lowest percentages of isolated pathogen. The isolated rhizosphere fungi were identified as; Chaetomium globosum, Aspergillus niger, Gliocladium virens, T. album, T. harzianum, T. viride and T. hamatum, recording frequencies of isolation of; 8%, 14%, 21%, 34%, 58%, 48% and 40%, respectively. Trichoderma spp. were selected for further study as being known to be effective antagonists, whereas, the remaining isolates were neglected.

Pathogenicity assay
Results demonstrated that the antagonistic potency of R. solani, M. phaseolina, F. oxysporum and F. solani were detected after 90 d from inoculation. R. solani recorded the highest disease rating scale and DSI of root rot of date palm (5-64.67%), followed by M. phaseolina (4-55.67%), respectively.
Similarly, F. oxysporum caused high incidence of root rot symptoms on the date palm plants (45.33%), and disease rating scale of (3). However, F. solani showed the lowest DSI of (36.33%) for root rot diseases and recorded disease rating scale of (3) ( Table 2). These variations among pathogens might be due to different root exudates produced by date palm which cause pronounced increase in the number of saprophytes around roots, thus will protect these roots from infection by soilborne fungal pathogens (Mogle and Mane, 2010;Xue Jing et al., 2011). In addition to presence of different resistance genes in roots of date palm plants accordingly expressing different responses against these pathogens.

In vitro antagonistic potential of the bioagents on the radial growth of the mycopathogens
A variation in in vitro antagonism between T. harzianum and the mycopathogens in concern was observed. The highest % of decrease in radial growth of F. solani, F. oxysporum, M. phaseolina and R. solani on PDA was recorded by T. harzianum; 87.10, 81.55, 68.55, and 77.6 %, respectively. However, T. hamatum expressed least antagonistic potential compared to those produced by the remaining bioagents. Moderate antagonistic potency was demonstrated by Bio-zeid and Plant guard commercial preparations as clear in Table (3).
The growth inhibitory effects of antagonists are in agreement with Rudresh et al., (2005) who observed 72.1 and 77.0% in vitro growth inhibition in R. solani and F. oxysporum, respectively, by T. harzianum and T. viride which also exhibited strong mycoparasitic activity and completely overgrew the pathogens mycelia once in contact with them. In addition, these observed results might be because different pathogens have different own defense mechanisms against enzymes and toxic substances produced by the different bioagents (Ahmed, 2013). Trichoderma secretes some chemical metabolites such as phenols, steroids, flavonoids, quinines, terpenoids, xantones, peptides, cytocatalasins, alkaloids and phenyl propanoids which might be responsible for their in vitro inhibitory or antagonistic activity (Muthu et al., 2006). Moreover, Trichoderma spp. antagonise the mycopathogens through mycoparasitism as they degrade their cell wall by producing lytic enzymes such as; chitinases, peroxidase, and glucan 1-3 B-glucosidases as stated previously by Mausam et. al., (2007); Ahmed, (2013), or through production of viridian mycotoxin by T. viride (Mishra, 2010;Wahyudi et al., 2011).

Effects of bioagents on root rot diseases severity of date palm under field conditions
As observed in Table (4), all tested bioagents significantly reduced severity of root rot diseases, compared with positive control soil infested with the concerned mycopathogens only. Moreover, they increased the percentage of survived date palm plants in both seasons of 2016 and 2017. However, different bioagents varied in their potential against disease severity. T. harzianum was most effective bioagent in controlling root rot diseases of date palm causing reduction in disease severity percentages to be (3, 2%), followed by T. viride (4.5, 3%), during the two growing seasons, respectively, compared with the positive control soil (17.0, 15.0%). On the contrary, T. hamatum demonstrated the lowest efficacy in controlling both diseases during both seasons recording; 10, 8.4%, respectively. The two commercial preparations expressed moderate antifungal potential by reducing disease severity to (6, 5.4%) for Bio-zeid, and (5.5, 4.5%) for Plant guard. These results were in agreement with Mwangi et al., (2011) who reported the ability of an isolate of T. harzianum (P52) to enhance the growth and control root rot disease caused by F. oxysporum f.sp. lycopersici on tomato seedlings. Different Trichoderma spp. is well known to antagonise many pathogenic fungi recording success in a number of crop diseases (Omomowoa et al., 2018).
These results might be explained according to the dual effect of bioagents which produce plant growth regulators (Karlidag et al., 2010;Wahyudi et al., 2011;Al-Rajhi, 2013), in addition to the chemical effect of their antioxidants that play a key role in improving plant physiology and metabolism (Al-Taweil et al., 2009;Wahyudi et al., 2011).
Trichoderma spp. are known to act as effective bioagents through different mechanisms such as; mycoparasitic activity (Matei and Matei, 2008), through production of antifungal enzymes such as Endo-chitinase, β-glucosidase and α-1,3-glucanase; through production of mycotoxins such as trichodermin (Balode, 2010), however, Trichoderma isolates may also compete for space and nutrients (Baset et al., 2010). number/ leaf. T. harzianum, T. viride, Plant guard and Bio-zeid, respectively, recorded the highest enhancement of all growth parameters compared with control treatment . On the other hand, T. album and T. hamatum gave the lowest increase in growth parameters during the two growing seasons of 2016 and 2017, respectively. These results were in agreement with those reported by (Khandelwal et al., 2012;Babu and Pallavi, 2013), who mentioned that Trichoderma spp. caused conspicuous improvement in the aforementioned crop growth parameters during the growing seasons. Trichoderma spp. are well known not only of being active bioagents for disease control, but act also as biofertilizers that enhance the growth of the plant through providing the soil with nutrients in utilizable forms that are absorbed by the plant roots, causing thus better plant nutrition (Pathak et al., 2007). In addition to the production of hormone like metabolites and release of nutrients from soil or organic matter thereby facilitate better plant growth (Yobo et al. 2010). Moreover, these results of enhancement of growth parameters might be due to variation in genetic pool of the date palm cultivars and/or the effects of climatic seasonal changes on their vegetative growth. Similar results were reported by Ahmed and Shaheen, (2016).

-Conclusion
Several bioagents such as T. album, T. hamatum, T. harzianum and T. viride were isolated from the rhizosphere of date palm trees, and they showed high in vitro and in vivo efficacy in antagonising certain soilborne fungal pathogens causing root rot diseases of date palm. In addition, they increased the growth parameters of date palm plants under field conditions. However, T. harzianum and T. viride were the best effective bioagents. These two isolates could thus be formulated in an economic and utilizable form to be applied as biofungicides to biocontrol soilborne mycopathogens, and as biofertilizers to enhance the growth of date palm in the field. Thus we could displace using the non-ecofriendly synthetic fungicides.