Evaluation of Endophytic Microorganisms (Trichoderma hazianum and Bacillus subtilus) as Biofungicides against Bayoud Disease (Fusarium oxysporum f. sp. albedinis) on Date Palm (Phoenix dactylifera L.)

The phoeniciculture is an essential element in the agro-economic development of oases (Bouguedoura et al., 2015; Vinita and Punia, 2016). The proposal of new management method using biotechnological procedures like plant breeding, phylogenic selection, micropropagation  etc.), has become more than necessary for the future of this field (Sedra, 2003). But all these methods have limits against bayoud disease on date palm culture (Djerbi, 2003). Bayoud disease, is a Fusarium wilt caused by Fusarium oxysporum f. sp. albedinis (Foa), is considered as the most destructive fungal disease in Moroccan and Algerian palm groves cultivated with important economical varieties like Bouskri, Majhoul and Boufeggous in Morocco and Deglet Nour and Feggous in Algeria, but all are susceptible to this parasite (Sedra 2005a,b; Djerbi, 2003). It’s originated from Morocco and its progression continues to advance from the west and central of Algerian palm groves (Benzohra et al., 2015; 2017). The losses reported by Djerbi (2003), estimated at more than 3 million palms are in a clear evolution, especially since several unaffected regions in the past are reconsidered as contaminated by Bayoud. This disease represents not only the risk to eliminate the best commercial varieties, but also to the phenomenon of desertification and the immigration of oasis habitants to large urban cities (Bouguedoura et al., 2015). The proposal used of prophylactic measures against this disease doesn’t seem able to stop its progression, especially since the epidemiological data are favored by a progressive spread of this disease, but these measures are important for southern Algeriangroves when the Bayoud is absent (Benzohra et al., 2017). Other method was used, a genetic control by resistant varieties is therefore another promising way to reduce this constraint, but unfortunately all resistant cultivars haven’t high food quality in date fruits (Sedra, 2005b).

To control and limit this constraint, especially in contaminated groves which contain the susceptible varieties, the biological control through the use of antagonistic microorganisms and mycorrhizal species remains the best way to combat this threat in these palm groves (El-Hassni et al., 2007; Jaiti et al., 2006; Jaiti et al., 2008; Dihazi et al., 2012; Benzohra and Megateli, 2017; Belaidi et al., 2020).

The objective of this work is to evaluate the capacity of in vivo effect in two antagonistic microorganisms Trichoderma harzianum and Bacillus subtilus on the incidence of bayoud disease by testing of three important commercial date palm varieties, but susceptible to Bayoud diseases (Deglet-Nour, Feggous and H-mira).

Date palm varieties
 
Three date palm varieties are used in the in vivo biocontrol test. These varieties are: Deglet-Nour, Feggous and H-mira (Fig 1). The Deglet-Nour is cultivated in the South-Central and East palm groves of Algeria and H-mira is cultivated in Saoura oases (South-West groves of Algeria).


 
Fungal material
 
The Foa isolate used in the inoculation test of date palm seedlings, was sampled from contaminated groves in Ghardaia province in 2016. The antagonistic microorganisms were obtained from soils of date palm rhizosphere of Bechar region in 2019 (Table 1). These strains of microorganisms used in this study provided by CRSTRA (Center for Scientific and Technical Research on Arid Regions, Biskra, Algeria), from laboratory of Plant Pathology and Protection, of Experimental Station of Biophysical Environment, Naama, Algeria. All isolates were conserved in PDA cultures (For 1000 ml : 200 g potato filtrate; 15 g glucose; 20 g agar-agar; pH=5,5), (Fig 1), with temperature of conservation at 04°C and incubation at 23°C (El-Hassni et al., 2007).


 
Preparation of inoculum
 
Date palm seedlings (2-3-months-old) were obtained by pre-germination of date palm seeds after their disinfection with Hypochlorite of Sodium at 2%, then rinsed with sterile distilled water three successive times (Sedra, 2005b). The date palm seeds are placed in Petri dishes containing the cotton soaked with sterile distilled water and then stored in the oven at a temperature of 23±2°C (El-Hassni et al., 2007). After 20 days of pre-germination, the seeds are transplanted into pots containing sterile peat for two months (Sedra, 1985).

The biocontrol test based on use of mixture ‘Foa inoculum + antagonistic’, this mixture is considered as treatment and control for plants treated with Foa inoculums alone taking account the same condition as above. Eight seedlings were used per treatment. After injection, seedlings were conserved in small greenhouse at 25±2°C with 16 h photoperiod.
 
Rating scale
 
Symptomsrating based on mortality rate of date palm seedlings. This rate mortality based on the calculation of dead plants compared to all the plants inoculated (Sedra, 2005b). The mortality rate (mr) formula is written as follows:     

 

 Statistical analysis
 
The experiments schemes were randomized complete blocks. Eight replicates (number of seedlings tested), per treatment were performed and each whole experiment was performed independently. All the collected data were submitted to ANOVA analysis using XLStat software 9 (AddinSoft, USA Ltd.) and the significance of differences among treatments was recorded at P<0.05. Multiple comparisons of the means were conducted according to the Newman-Keuls test at P<0.05.

The statistical analysis of results showed significant differences between the date palm varieties and also between two antagonistic microorganisms (Table 2). The results showed a high level of effectiveness of the two microorganisms on theincidence of Bayoud disease of date palm compared to the control. The control (direct inoculation with Foa alone), presented a very high mortality rate up to 100% (Table 2), with typical symptoms of bayoud disease by vascular wilt and discoloration (Fig 2). The antagonistic fungus T. harzianum showed the total absence of bayoud disease symptoms without mortality rate (rm) observed (0%), (Table 2). The second species, the bacterium B. subtilus showed an antagonistic behavior less than the fist species (T. harzianum), withrm value varied according to the date palm variety. In two varieties (Deglet-Nour and H-mira), B. subtilus showed the total absence also of typical symptoms of bayoud (0%), but in the variety Feggous, the rm values was 50%. In comparison between these two species, we can say that the fungus T. harzianum was more efficiency than the bacterium B. subtilus.





Our findings in this study show that the antagonistic microorganisms tested have significant biocontrol activity against Bayoud disease when tested by an in vivo biocontrol on bayoud disease incidence. These microorganisms are: one fungus named Trichoderma harzianum and one bacterium named Bacillus subtilus (Table 2), using screening test based on inoculation by Foa isolate of three important date palm varieties in Algeria (Deglet-Nour, Feggous and H-mira). The mortality rate varied significantly according to the type of antagonist. The absence of mortality rate was observed using T. harzianum, while the bacterium B. subtilus showed the efficiency two varieties (Deglet-Nour and H-mira), while the Feggous variety showed the mortality arrived at 50%. In comparison between these two antagonistic microorganisms, the fungus T. harzianum is more efficient than the bacterium B. subtilus.

Many reports from the world has been published about the importance of biological control as the best way to combat Fusarium wilt (Alabouvette, 2006; Sajeena et al., 2020; Cheng et al., 2019; Abed et al., 2016; Ou-Zine et al., 2020). This method of control can enhance the plant defense machanisms against Fusarium disease and also other phytopathogenic fungi (El-Hassni et al., 2007).

The importance of antagonistic fungi like Trichoderma harzianum as biofungicide against fusarium wilt has been reported by Kala et al., (2016), against Fusarium wilt of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris, by Sahampoor et al., (2020), on Fusarium wilt of cucurbits caused by Fusarium oxysporum f. sp. radicis-cucumerinum and by Fitrianingsih et al., (2019), on Fusarium wilt oftomato (Lycopersicum esculentum L.) caused by Fusarium oxysporum f. sp. radicis-lycopersisci.

The antagonistic bacterium Bacillus subtilus was also reported by many authors against Fusarium wilt diseases. Among these fusarium wilt, we can cite: Fusarium oxysporum f. sp. ciceris of chickpea (Cicer arietinum L.), (Zaim et al., 2013; 2018; Rouag et al., 2019; Suthar et al., 2019), Fusarium oxysporum f. sp. radicis-lycopersisci of tomato (Lycopersicum esculentum L.), (Estefania and Ligia, 2018) and Fusarium oxysporum f. sp. abedinis of date palm (Phoenix dactylifera L.), (El-Hassni et al., 2007).

In this study, these two antagonistic microorganisms (T. harzianum and B. subtilus), can enhance the ability to resist bayoud disease by the three date palm varieties used in the in vivo biocontrol test (Deglet-Nour, Feggous and H-mira).

This work had the objective to in vivo biocontrol based on the use of two antagonistic microorganisms, one fungus speciesTrichoderma harzianum and one bacterium Bacillus subtilus, against Bayoud disease of date palm caused by the soil fungus Fusarium oxysporum f. sp. albedinis (Foa), on three date palm varieties (Deglet-Nour, Feggous and H-mira). The results of this study showed significant differences between the two antagonistic microorganisms as well as the control (inoculation with Foa strain inoculum). If we make a comparison between two antagonistic species, we can say that T. harzianum is more efficient than that species B. subtilus, rm% values equal to 0% for the first, 50 for the second, respectively.

In conclusion, the use of biological agents like antagonistic microorganisms to enhance the resistance in the susceptible varieties against this pathogen represents an ecologically friendly alternative to pesticides repeatedly used to control this kind of plant diseases. This strategy has more significance against soil-borne pathogens such as bayoud disease on date palm in our contaminated palm groves, especially in south-west region of Algeria, for fighting strategies are very restricted or no large used by our farmers.

The funding of this research was by FNR project (BayoudBiol - Div. Phoeniciculture - 2021-2024), of CRSTRA research centre (Centre for Scientific and Technical Research on Arid Regions, Biskra, Algeria) and DGRSDT (Directorate General for Scientific Research and Technological Development, Algiers, Algeria).

All authors declare that they have no conflicts of interest.