Induction of Systemic Resistance in Sugar- Beet against Root-Knot Nematode with Commercial Products

Sugar-beet (Beta vulgaris L.) is considered an important root crop, which is ranked second to sugar-cane for supporting the expansion of Egyptian sugar industry. Root-knot nematodes (RKNs) Meloidogyne spp. are among the most deleterious plant pathogens since these organisms play a detectable role in limiting the productivity of such economic agriculture crop. The root –knot nematode Meloidogyne incognita (Kofoid & White) Chitwood is among the most important nematode infesting sugar-beet. Many efforts to protect such crop from root-knot nematodes infestation are crucial. Because of the lack of plant resistance to most species of root-knot nematode as well as the environmental restrictions on nematicidal use for controlling plant parasitic nematodes, biological control and other eco-friendly disease control measures have gained recently increasing interest. The activation of the plant's own defense system through biotic and abiotic agents, called elicitors, has been considered as a focus of research only in recent years for the control of plant pathogens. The resulting elevated resistance due to an inducing agent upon infection by a pathogen is called induced systemic resistance (ISR) or systemic acquired resistance (SAR) [1].


Introduction
Sugar-beet (Beta vulgaris L.) is considered an important root crop, which is ranked second to sugar-cane for supporting the expansion of Egyptian sugar industry. Root-knot nematodes (RKNs) Meloidogyne spp. are among the most deleterious plant pathogens since these organisms play a detectable role in limiting the productivity of such economic agriculture crop. The root -knot nematode Meloidogyne incognita (Kofoid & White) Chitwood is among the most important nematode infesting sugar-beet. Many efforts to protect such crop from root-knot nematodes infestation are crucial. Because of the lack of plant resistance to most species of root-knot nematode as well as the environmental restrictions on nematicidal use for controlling plant parasitic nematodes, biological control and other eco-friendly disease control measures have gained recently increasing interest. The activation of the plant's own defense system through biotic and abiotic agents, called elicitors, has been considered as a focus of research only in recent years for the control of plant pathogens. The resulting elevated resistance due to an inducing agent upon infection by a pathogen is called induced systemic resistance (ISR) or systemic acquired resistance (SAR) [1].
However, induced resistance to plant parasitic nematodes has not been as extensively studied as that of fungi and bacteria. Ibrahim, et al. [2] recorded the capability of humic acid as well as thiamine at the concentration of 2000 ppm to induce resistance in sugar-beet against M.incognita and increase the activity of polyphenol oxidase (PPO) and peroxide oxidase (PO) enzymes compared with non-infected plants. On the other hand, plant growth promoting rhizobacterium (PGPR) belonging to Bacillus spp. are being exploited commercially for plant protection to induce systemic resistance against various pests and pathogens. PGPR mediated rhizobacteria is often associated with the onset of defense mechanisms by expression of various defense related enzymes such a glucanase, chitinase, phenylalanine ammonia lyase (PAL), peroxidase (PO), and polyphenol oxidase (PPO) and accumulation of phenols [3] .In this point of view, the present work was carried out in order to study the impact of promoting growth rhizobacterium (PGR), Bacillus megaterium, Nemastrol active ingredients extract as resistance inducers to sugar-beet plant infected with M. incognita under greenhouse conditions.

Materials and Methods
Two greenhouse experiments were conducted at Nematological Research Unit (NERU), using sandy and clayey soil in order to evaluate the nematicidal properties of the commercial formulation of rhizobacterium, Bacillus megaterium (Bio-arc), the commercial biocide, Nemastrol against the root-knot nematode, M. incognita and the resulting effect on plant growth parameters of sugar-beet var. Negma. Induced resistance (IR) of such bio-agents was assayed through chemical composition and enzyme activities.

Tested bio-agents
Bio-arc: A native commercial formulation of phosphorus soluble bacterium, Bacillus megaterium (25×10 6 cfu/g) @ 2.5 g/L of distilled water, was obtained from Agricultural Research Institute, Giza, Egypt and enrolled by the Ministry of Egyptian Agriculture under No. 1087.
Tested bio-agents rates: The tested bio-agent, Nemastrol was applied @ the rate of 0.25 ml/pot. However, Bio-arc was added using four different rates of 5, 10, 15 and 20 ml/pot in single application.
For each treatment, dry weight of shoot (1 g) was subjected to chemical analysis in order to evaluate total nitrogen, crude protein, total carbohydrate and total phenol. Samples of dried leaves were ground, wet digested and nitrogen (N), phosphorus (P), potassium (K) contents were determined according to kjeldahl methods [7] A.O.A.C. incognita and tested for enzymatic activities. The same protocol as outlined before was repeated. Roots were collected at different intervals (0, 3, 9 and 15 days after treatment and nematode inoculation) and assayed for activities of Peroxidase (PO) and Polyphenol Oxidase (PPO).
Preparation of enzyme extract: Enzyme extracts were prepared following the method described by [3] Maxwell and Bateman (1967(. Dry root tissues (0.5 g) of each treatment were ground in 3 ml Naphosphate buffer at pH 6.8 in a mortar and then centrifuged at 1.500 g/20 min at 6°C. The resultant supernatant fluids were processed for enzyme assays.
Peroxidase activity (PO): Peroxidase was assayed using photochemical method as described by [11] Amako et al. The reaction mixture was added as the following sequences, 1500 ml phosphate buffer., 480 ml hydrogen peroxidase., 1000 ml pyrogallol, 20 ml sample extract. The increasing in the absorbance at 430 nm was recorded against blank with phosphate buffer instead of enzyme extract. One unit of enzyme activity was defined as the amount of the enzyme, which changing the optical density at 430 nm per min. at 25°C under standard assay conditions. Specific activity was expressed in units by dividing it to mg protein.

Polyphenol oxidase (PPO):
Polyphenol oxidase was assayed using photochemical method as described by Coseteng and Lee [12]. The reaction mixture was added as the following sequences: 2.7 ml potassium phosphate buffer 90.05 M, pH 6.2, 0.25 ml of 0.25 M catechol, 0.05 ml of enzyme extract. The increasing in absorbance at 420 nm was measured. One unit of enzyme activity is defined as the amount of the enzyme that causes an increase of 0.001 absorbance unit per minute at 25°C.

Data analysis:
Statistically, the obtained data were subjected to analysis of variance (ANOVA) [13] (Gomez and Gomez) followed by Duncan's multiple range tests to compare means [14].

Results
The influence of the two bio-control agents namely Bio-arc (a commercial formulation of B. megaterium) at four tested rates (5, 10, 15, and 20 ml) and Nemastrol @ 0.25 ml singly and concomitantly on plant growth response of sugar-beet plant var. Negma infected with M. incognita and grown in two soil types i.e. clayey and sandy is summarized Table 1. (Results revealed that M. incognita infection caused a significant reduction in plant growth parameters (shoot and root length, shoot weight) with reduction percentage in total plant fresh weight reached 35.0 and 64.0% in clayey and sandy soil respectively. Irrespective to soil type and tested rates, all treatments showed remarkable increase in plant growth parameters in terms of shoot length, shoot and root weight with various degrees. In single application, it was evident that the effectiveness of bio-arc to enhance plant growth parameters increased with the increase of addition in the two soil types. Plant growth response of sugar-beet infected with M. incognita was pronounced in sandy soil more than clayey soil. In sandy soil, a significant improvement in shoot length (85.8%), plant fresh weight (174.1%) and shoot dry weight (350.0%) was recorded with Bioarc @ the rate of 20 ml/plant. Similar trend was noticed with sugar-beet grown in clayey soil with percentage of increase in shoot length, total plant fresh weight and dry shoot weight reached 70.4, 41.7 and 180.0%, respectively. However, Nemastrol at the rate of 0.25 ml resulted a pronounced improvement in plant growth parameters in terms of shoot length (100.0, 10.8%), total plant fresh weight (70.5, 44.4%) and shoot dry weight (200.0, 150.0%) of sugar-beet grown in clayey and sandy   soil respectively. In concomitant treatment, Bio-arc (20 ml)+Nemastrol (0.25 ml) was the best and showed significant improvement in plant growth parameters in terms of shoot length (92.6, 127.5%) and total plant fresh weight (91.7, 370.4) of sugar-beet grown either in clayey or sandy soil, respectively. Oxamyl as a standard nematicide showed moderate improvement in pervious criteria of sugar-beet grown in clayey soil with increase percentages 39.3, and 47.4 respectively. Similar trend was noticed with shoot length (54.2%) and dry shoot weight (50%) of sugar-beet grown in sandy soil treated with oxamyl.
Regarding the impact of Bio-arc and Nemastrol singly and concomitantly on the development and reproduction of the rootknot nematode, M. incognita infecting sugar-beet grown in clayey and sandy soil is documented ( Table 2). Irrespective to soil type and tested rates results revealed that total nematode population was significantly suppressed with all tested treatments with reproduction factor ranged from 1.4 to 11.6 in clayey soil and from 0.8 to 18.6 in sandy soil compared to inoculated plants (Rf=30.0, 35.9) respectively. Among tested treatments, Nemastrol significantly suppressed total nematode population (RF=1.9, 2.2), root galling (RGI=3.0, 3.0), number of egg masses (EI=3.0, 3.0) and number of eggs /10 egg masses (Red. %=76.5, 74.6) in clayey and sandy soil, respectively. However, concomitant treatment showed better results than did bio-arc alone at four tested rates. Among the concomitant treatment the greatest reduction in total nematode population was recorded in clayey and sandy soil which received the dual application of Bio-arc (20 ml) and Nemastrol (0.25 ml) with reproduction factor 2.2, 2.6 and reduction percentages reached 92.8, 92.6%. Meanwhile, total nematode population was significantly suppressed with oxamyl introduced to clayey soil (Rf=1.4) and sandy soil (Rf=0.8) relative to control plants where Rf=30.0 and 35.9, respectively. Nevertheless, number of eggs/ egg mass were significantly suppressed with oxamyl application with percentage of reduction amounted to 76.7 and 74.5% in clayey and sandy soil respectively.

Biochemicals activities
Nitrogen, phosphorus and potassium contents: NPK contents were significantly suppressed due to nematode infection with reduction percentages 34.1, 35.5 and 39.2% in clayey soil and 32.2, 37.5 and 39.1% in sandy soil. However, a remarkable induction in NPK content was recorded with the application of Bio-arc + Nemastrol (20 ml+0.25 ml) with % of increase amounted to 34.94, 41.40, 48.43 and 39.55, 45.0, 48.99 in clayey and sandy soil respectively (Figures 1-3).
Total chlorophyll content: Chlorophyll a and b were moderately affected due to nematode infection with reduction % in total chlorophyll reached 32.5 and 32.3% in clayey and sandy soil respectively. Application of such treatments revealed a considerable induction with the dual application of Bio + Nemastrol (20 ml+ 0.25 ml) and oxamyl as well with % of increased reached 37.0, 42.6 and 38.6, 40.9% in clayey and sandy soil consecutively (Figure 4).   Phenol content: The total phenol evaluated in leaves of sugar-beet infected with M. incognita revealed a moderate enhancement compared to control plants. However, phenol content showed different degrees of reduction in all treatments compared to untreated uninoculated plants grown in clayey and sandy soil. (Figure 7) Defense related proteins: The tested materials viz. Bio-arc (20 ml), Nemastrol (0.25 ml), Bio-arc+Nemastrol (20 ml+0.25 ml) and oxamyl as well differed in their ability to stimulate Peroxidase (PO) and Polyphenol Oxidase (PPO) activities in sugar-beet plant inoculated with M.incognita (Figure 8). In untreated uninoculated plants, the activities of PO and PPO remained higher and attained their peak at the 9 th day and thereafter a decline was noticed at 15 th day. On the other hand, the least induction of PO and PPO was recorded with plants untreated and inoculated with nematodes and showed slight decline 3 days after nematode inoculation then increased and reached their peak at 9 th day. However, increased PO and PPO activities were more pronounced in Bio-arc+Nemastrol (20 ml+0.25 ml) followed by oxamyl then Nemastrol compared to untreated inoculated plants. In such treatments, the dual application Bio-arc + Nemastrol performed the best since PO& PPO activities were increased and reached their peak at the 3 rd day after nematode inoculation then declined at 9 th day followed by slightly increment at 15 th day . Meanwhile, the increased activity of PO &PPO remained higher in plants treated with oxamyl and reached their peak at 9th day after nematode inoculation.

Discussion
Induction of systemic resistance (ISR) of plants against pathogens is a widespread phenomenon that has been intensively investigated in fungi and bacteria with respect to its potential use in plant protection. However, little attention has been given to nematode pests. Acquired or induced resistance can be achieved by inoculating a plant with incompatible or weak pathogens or by applying biotic or abiotic inducers [15]. The root-knot nematode, M. incognita caused a significant reduction in plant growth parameters (shoot and root length, shoot weight) with reduction percentage in total plant fresh weight reached 35.0 and 64.0% in clayey and sandy soil respectively. Application of   phosphorus solubilizing bacterium (PSB), B. megaterium singly or concomitantly with Nemastrol has potential as a promising biocontrol candidate against root-knot nematode, M. incognita infecting sugarbeet var. Negma. As for single application, the effectiveness of Bio-arc to enhance plant growth parameters increased with rates increase in the two soil types.
Plant growth response of sugar-beet infected with M. incognita was more pronounced in sandy soil than clayey soil. In sandy soil, a significant improvement in shoot length, plant fresh weight and shoot dry weight was recorded with Bio-arc @ the rate of 20 ml/plant. This result support the findings of El-Deriny and Ibrahim [16,17]. However, in concomitant treatment, Bio-arc+Nemastrol (20 ml+0.25 ml) performed the best and showed significant improvement in plant growth parameters in terms of shoot length (92.6; 127.5%) and total plant fresh weight (91.7; 370.4%) of sugar-beet grown either in clayey or sandy soil. The presence of cytokinins in Nemastrol suggests a dynamic role for lateral root development. Irrespective to soil type and rates of application, total nematode population, root galling, number of egg masses and number of eggs/ egg mass were significantly suppressed with all treatments of Bio-arc and/or Nemastrol. The phosphate solubilizing bacterium (PSB) B. megaterium is considered a microorganism capable of dissolving the unavailable phosphorus compounds in soil rendering them available for growing crops [18]. Increased phosphorus concentration may lead to reduction in root-knot nematodes. B. megaterium has been evaluated for their effects on a variety of root-knot nematodes [16,17,19,20] reported that B. megaterium greatly reduced numbers of galls, females and egg masses of M. incognita in the roots of sugar-beet followed by B.subtilis, Paecilomyces lilacinus, P. fumosoreus and Trichoderma album respectively. Furthermore, B. megaterium can extensively colonize the rhizosphere and reduce the sugar-beet cyst nematode infection under greenhouse trials [21]. B. megaterium produce antibiotic compounds [22] although no compounds from B. megaterium have been reported with activity against nematodes.
Nevertheless, Nemastrol performed the best and significantly suppressed total nematode population; root galling, number of egg masses and number of eggs /egg mass in clayey and sandy soil. The suppressive effect of such product could be attributed to the presence of mixture of enzymes i.e. chitinase and glucanase that dissolve chitin of nematode egg shell. However, concomitant treatment using Nemastrol+ Bio-arc showed better results than did Bio-arc alone at four tested rates. The greatest reduction in total nematode population was recorded with clayey and sandy soil receiving the dual application of Bio-arc (20 The impact of screened treatments on chemical components viz. NPK, chlorophyll, total carbohydrates, crude protein and total phenol in sugar-beet leaves infected with M. incognita revealed a remarkable induction in chemical constituents with the application of Bio-arc+Nemastrol (20 ml+0.25 ml). Conversely, the highest increase in total phenol percentage was recorded with untreated inoculated plants as a hypersensitive reaction (HR) to nematode infection. Plants are endowed with defense genes which are quiescent in healthy plants. When these genes are activated with various factors they induce systemic resistance against disease. Rhizobaceria induce systemic resistance by activation of various defense-related enzymes viz. PO, PPO and PAL. Recently, research work has demonstrated that the bio-agent Pseudomonas fluorescens might stimulate the production of biochemical compounds associated with the host defense [23]. Of these, early induction of peroxidase is more important as it is the first enzyme in the phenylpropanoid pathway, which leads to production of phytoalexin and phenolic substances leading the formation of lignin [24]. Conspicuously, the current investigation recorded the higher activity of Peroxidase in plants treated with Bio-arc+Nemastrol ( 20 ml+0.25 ml) and reached its peak at 9 days generating the speculation of induced defense responses in sugar-beet infected with M. incognita. Peroxidase activity in roots is important in the reinforcement of cell walls at the border of infection in resistant plants and that are considered as important components of active defense response of nematode invaded tissue [25]. The trend of increasing PPO activity was similar to that of PO in all treatments. Increased activity of Peroxidase (PO) or Polyphenol Oxidase (PPO) has been elicited by biocontrol agent strains in different plants [26,27]. Finally it can be concluded that use of such inducers viz. Bio-arc and Nemastrol singly and concomitantly represent a promising new approach for the control of the target root-knot nematode, M. incognita infecting sugar-beet within an environmental friendly integrated pest management via enhancing the resistance of plant to nematode.