Potential of Spirulina platensis and Saccharomyces cerevisiae in Integration with Organic Amendments Towards Root–Knot Nematode, Meloidogyne incognita on Soybean

A greenhouse experiment was carried out to assess the potential of the cyanobacterium, Spirulina platensis , yeast, Saccharomyces cerevisiae and two organic amendments namely, molasses and yeast extract singly or integrated for the management of root-knot nematode, Meloidogyne incognita on soybean . Results revealed that soil drenched with S. cerevisiae outperformed S. platensis in promoting plant criteria i.e. plant length, shoot and root weights and total plant fresh weight and reducing nematode parameters in terms of total nematode population and root galling. A synergistic impact on plant and nematode characters resulted in the combination of S. cerevisiae and S. platensis . However, the beneficial effect of both S. platensis and S. cerevisiae increased with the introduction of molasses to soil recording 96.6, 91.3 and 92.3% reduction in nematode population, number of females and galls, respectively as well as 14.8 and 261.1% improvement in plant length and total plant fresh weight, respectively . Meanwhile, integration of cyanobacterium with organic amendments (yeast extract and molasses) recorded the highest significant (P≤0.05) values of chlorophyll A and B, nitrogen, phosphorus and potassium. The present findings suggest that soil application with both S. platensis and S. cerevisiae as well as organic amendments could be environmental safe, cost-effective and promising alternative of nematicides against M.incognita.


INTRODUCTION
Soybean (Glycine max L.) is a vital globally leguminous crop and a major source of oil and protein which comprise approximately 20 and 40% of the soybean, respectively (Clemente and Cahoon, 2009). Plant parasitic nematodes are among the most important pests of soybean causing an estimated annual crop loss of $78 billion worldwide (Lima et al., 2017) and an average crop yield loss of 10-15%. Root-knot nematodes, Meloidogyne spp., particularly M. incognita are amongst the most important widespread pathogens and consistent threat to soybean production that have been emerged in recent years (Roth et al., 2020). A total of 75% yield loss has been reported on early-maturing soybean in a field infested with M. incognita (Emerson et al., 2018). Biological control using bacteria and fungi is considered to be an efficient alternative method that can be combined with others within an integrated management system as aim to decrease the negative environmental impacts of the excessive nematicides. During the last decade, great interest has been paid to the use of cyanobacterium and yeast as bio fertilizers in agriculture because of their bioactivity and safety for human and the environment. Cyanobacterium, Spirulina platensis belonging to Cyanophyta is a blue-green alga with medicinal and nutritional interest due to its richness in proteins, minerals, carbohydrates, phytopigments and antioxidants (Bitam and Aissaoui, 2020).
Extracts and exudates of cyanobacterium have been reported to inhibit egg hatching and cause immobility and mortality of 2 nd juveniles of plant parasitic nematodes in vitro (Holajjer et al., 2013). Various studies have shown the nematicidal potential of S. platensis against root-knot nematode (Shawky et al., 2009;Sharaf et al., 2016;Hamouda et al., 2019;El-Eslamboly et al., 2019). Yeast, Saccharomyces cerevisiae is a promising plant growth promoter for different crops as well as a biocontrol agent of root-knot nematodes (Karajeh 2013;Mokbel and Alharbi 2014;Youssef and El-Nagdi, 2018). Yeast increases plant resistance against harmful pests, especially parasitic nematodes, by increasing the concentration of total phenolic compounds in the roots (Karajeh, 2013). Hamouda et al. (2019) revealed that the usage of S. cerevisiae and S. platensis in combined treatment revealed the greater antagonistic action on M. incognita in potted banana. The addition of organic amendments may improve soil structure and fertility, so that plant vigor improves and plants are better able to tolerate the effects of nematodes (Stirling, 1991;El-Nagdi et al., 2019), but direct effects on nematodes may occur through the release of toxic compounds during decomposition, stimulation of natural enemies or through changes in the level of plant resistance. Sugarcane molasses as organic amendment was found effective to manage root-knot nematodes (Khan et al., 2016). The present study was carried out to investigate the effectiveness of S. platensis and S. cerevisiae and two organic amendments, molasses and yeast extract on M. incognita and plant growth of soybean under greenhouse conditions.

MATERIALS AND METHODS
A greenhouse experiment was conducted at the Nematological Research Unit (NERU), Faculty of Agriculture, Mansoura University, Egypt, in order to evaluate the nematicidal properties of cyanobacterium (blue green alga), Spirulina platensis, yeast, Saccharomyces cerevisiae as well as yeast extract and molasses as organic amendments against M. incognita infecting soybean.

Microbial and organic agents
All tested agents except yeast were obtained from Microbiology Dept., Fac. Agriculture, Mansoura Univ., Mansoura, Egypt.

Cyanobacterium, Spirulina platensis
Aqueous extract of the blue green alga, Spirulina platensis was prepared and concentrations of 50 and 100 ppm were done.

Yeast, Saccharomyces cerevisiae
Dried powder of yeast, Saccharomyces cerevisiae was purchased from a local shop and a total of 0.5g of yeast was dissolved in 100 ml of distilled water and concentrations of 50 and 100 ppm were prepared.

Organic amendments
Yeast extract powder and molasses both were obtained from Microbiology Dept., Fac. Agriculture, Mansoura Univ., Mansoura, Egypt.

Yeast extract
A total of 0.5g of yeast extract powder was dissolved in 100 ml of distilled water and concentrations of 50 and 100 ppm were prepared.

Molasses
A total of 0.5 g of molasses mixed with 100 ml distilled water and concentrations of 50 and 100 ppm were prepared.

Experimental design
Soybean seeds cv. Giza111 were sown, three seeds per hole, in plastic pots (15 cm-d) containing sterilized sandy loam soil (1:1). Two weeks later, seedlings were thinned to one plant per pot and inoculated with 600 second stage juveniles (J2s) of M. incognita spread around the base of plant and as close to the roots as possible. Simultaneously, cyanobacterium (S. platensis), yeast (S. cerevisiae) as well as molasses and yeast extract as organic amendments were applied as soil drench singly and in combination at a rate of 4ml / pot. Plants free of nematodes and treatments were served as healthy plants (Check). While those receiving nematode inoculum were served as inoculated untreated plants (Control). Two days after nematode inoculation the conventional nematicide, oxamyl was applied to soil at the rate of 0.3ml/pot. Treatments were as follows: 1) Cyanobacterium; 2) Yeast; 3) Molasses; 4) Yeast extract; 5) Cyanobacterium + yeast; 6) Cyanobacterium + molasses; 7) Cyanobacterium + yeast extract; 8) Yeast + molasses; 9) Yeast extract + molasses; 10) Cyanobacterium + yeast + molasses; 11) Cyanobacterium + yeast extract + molasses; 12) Oxamyl 24% L; 13) Plant free of nematode, and 14) Nematode only.
Pots were set up in a completely randomized design with four replicates for each treatment. Pots were irrigated with tap water as needed. Plants were harvested 45 days after nematode inoculation. Data dealing with shoot and root fresh weights, dry shoot weight, shoot and root lengths, were recorded. Second stage juveniles (J2s) were extracted from 250g soil using sieving and modified Baermann technique (Goodey, 1957). The nematode suspensions were examined in a Hawksely counting slide with an anatomy microscope to quantify the numbers of juveniles. Roots were stained with acid fuchsin in lactic acid (Bybd et al., 1983) and numbers of galls, developmental stages and females were determined.

Biochemical tests
At the end of experiment, dried leaves of soybean were subjected to biochemical tests. Chlorophyll A and B, were estimated according to the method of Bradford (1976) while Nitrogen (N), phosphorus (P) and potassium (K), were estimated using the method described by Malik and Singh (1980).

Data analysis
Data of the current investigation were subjected to statistical analysis of variance (ANOVA) (Gomez and Gomez, 1984) using CoStat computer software (CoStat, 2005), and means were compared according to Duncan's multiple range test at P≤0.05 (Duncan, 1955). Table (1) represent the impact of cyanobacterium, yeast, molasses and yeast extract applied singly or in integration on M. incognita infecting soybean Giza 111 under greenhouse conditions. Results indicated that M. incognita infection caused a Egypt. J. Agronematol., Vol. 21, No.2 (2022)  significant decrement in plant growth parameters with reduction percent in plant length and shoot weight reached 30.19 and 53.54%, respectively. Yeast, S. cerevisiae stimulated soybean plant criteria in terms of shoot fresh weight (69.57%) and total plant fresh weight (81.48%) better than aqueous extract of cyanobacterium, S. platensis. Hence, in dual treatment, integration of both yeast and cyanobacterium showed a synergistic impact on such parameters recording 82.61and 98.15%, respectively.

Data in
Notably, single treatment of organic amendment namely, yeast extract showed the best augmentation in shoot weight and plant fresh weight with percentages of increase amounted to 132.61 and 146.30, respectively. However, integration of molasses with cyanobacterium significantly increased the effectiveness of S. platensis on soybean plant fresh weight (170.37%) better than yeast extract addition (144.44%). Similar trend was noticed with shoot dry weight. Apparently, triple applications gave better results than did single or dual ones. Hence, combined treatment of molasses with both S. platensis and S. cerevisiae gave the best augmentation in total plant length (14.8%), shoot weight (258.7%) and total plant weight (261.1%). Conversely, soil treated with the aqueous extract of the single microbial agent, S. platensis and two organic amendments gave a non-significant increase in such parameters. Oxamyl recorded the best result (32.6%) in improving plant length only followed by molasses (10.1%). Among the observed data, shoot dry weight of soybean was not significantly (P≤0.05) increased compared to positive and negative control.
The levels of N, P, K and chlorophyll in leaves of soybean infected with M. incognita varied significantly among the studied treatments (Table 2). Control treatment recorded the lowest values of chlorophyll A, B and NPK. However, irrespective to oxamyl, integration of cyanobacterium with yeast extract and molasses recorded the highest significant values of chlorophyll A (0.69); chlorophyll B (0.40), N (4.11), P (0.365) and K (2.75) with percentages of increase amounted to 24.17, 18.89 and 24.43% (Fig.1, ae).  The role of tested bio-agents in reducing development and reproduction of M. incognita infecting soybeans was accentuated in Table (3) and Fig. (2). Untreated plants infected with M. incognita harbored higher number of nematode population, where RF=1.49. All treatments resulted in a remarkable suppression in nematode population densities compared to control. The highest percentage of reduction in nematode parameters was obtained with oxamyl (100%). Yeast, S. cerevisiae significantly suppressed numbers of total nematode population (55.0%), females (66.7%) and galls (69.2%) better than S. platensis recording reproduction factor (RF) equal to 0.70.  However, an antagonistic properties were recovered with the integration of both microbial bio-agents; yeast + cyanobacterium against such nematode parameters scoring 73.7, 75.0 and 76.9%, respectively recording RF= 0.40 compared to individual applications.

DISCUSSION
Several approaches including biological control and organic amendments have been launched with different degrees of success in the management of root-knot nematodes associated with soybean (Oyekanmi and Fawole, 2010). Yeast and cyanobacterium both are considered as new promising alternatives of chemical fertilizers and nematicides. Yeast, S. cerevisiae led to a significant decrease in the numbers of rootknot nematodes infecting beans (Osman et al., 2020) and cucumber (Karajeh, 2013). Its contents of carbohydrates produce ethanol and carbon dioxide, which have toxic effects on nematodes. Besides, yeast causes increments of plant resistance, as it competes with nematodes for nutrients and plays a role for making the physical and chemical properties of the soil unsuitable for the activity of plant parasitic nematodes (El-Nuby, 2021). The previous results confirmed the present findings in which the individual treatment of S. cerevisiae stimulated soybean plant criteria in terms of shoot fresh weight and total plant fresh weight, and significantly induced the highest reduction percentages in nematode criteria in terms of number of total nematode population, females and number of galls. Treatment of S. cerevisiae increased nitrogen, phosphorus potassium and the photosynthetic pigments (chlorophyll A, B) in soybean plant. These results confirmed the findings of Moller et al. (2016) who reported that yeast produces many active natural ingredients such as enzymes, plant hormones and oxen's groups (indole-3-acetic acid, IAA), which have an effective impact on increasing the productivity of the plant and the proportions of major elements in the leaves. The effectiveness of cyanobacterium, S. platensis was also expressed by suppressing root galling and final nematode population as well as improving vegetative and root growth of soybean. The nematicidal potential of such microbial agent is likely due to the huge production of secondary metabolites having nematicidal properties such as phenols, antioxidants, polyphenols, and flavonoids (Shawky et al., 2009;Chtourou et al., 2015). The presence of hormone components i.e., indoles, cytokinins, gibberellins, brassinosteroids, and other plant growth regulators including amino acids, peptides, and polyamines has beneficial influence on plant productivity and proportions of major elements (Lee et al., 2008;El-Eslamboly et al., 2019).
Soil drench with both S. platensis and S. cerevisiae gave better performance in plant vigor and nematode control providing soybean with various compounds (phytohormones and proteins) which led to an increment in major elements (nitrogen, phosphorous, potassium) and the photosynthetic pigments. This finding was conformed with that of Hamouda et al., (2019) who reported that the usage of S. cerevisiae and S. platensis in combination treatment revealed the greater antagonistic action on M. incognita in potted banana. The presence of flavonoids, polyphenols, phenylalanine, and antioxidants in yeast and cyanobacterium algal extract may affect nematode penetration and reduce gall formation (Chtourou et al., 2015). The present study revealed that molasses and yeast extract both had a significant effect on the management of root-knot nematode, M. incognita and consequently soybean plant growth. The addition of molasses with S. platensis and S. cerevisiae as soil drench achieved the highest significant reduction in nematode population and root galling of M. incognita infecting soybean and improved plant growth and chemical constituents as well. As reported by Schenck (2001), molasses soil amendments may supply carbohydrates and alter the C/N ratio. This affects the soil microbial ecology, usually resulting in lowered populations of plant parasitic nematodes as well as having other favorable effects on plant growth. The same author added that molasses do not pose a threat to the environment that chemical pesticides do, since it is readily decomposed in soil to CO2 and harmless organic products. Also, its suppressant effect was probably due to antagonism by microorganisms, to changes in oxygen concentration due to microbial metabolism of molasses, or to the release of toxic compounds from decomposing molasses. The leaf nutrient concentrations i.e. NPK and chlorohyll A and B were significantly influenced by various biotic treatments and help in optimizing fertilizer scheduling (Pestana et al., 2005;Rodriguez-Navarro et al., 2011;Ortiz-Liebana et al., 2022) and reached their maximum values by the addition of S. platensis and yeast extract and molasses.

CONCLUSION
It can be concluded that S. platensis and S. cerevisiae as well as organic amendments (yeast extract or molasses) can be recommended as environmentally and friendly agents for the bio-management of root-knot nematode, M. incognita on soybean in organic farming.