The role of Bacillus megaterium and other bio-agents in controlling root-knot nematodes infecting sugar beet under field conditions

Abstract

A micro-plot field experiment was conducted in loamy soil naturally infested with Meloidogyne spp. to assess the potential of bio-agents namely bio-arc (Bacillus megaterium), nemastrol (a mixture of active ingredients), humisun (humic acid), and dried sweet basil callus to suppress nematodes’ population and induce resistance in sugar beet. Results indicated that integration of two or more components of such bio-agents gave better results in sugar beet growth parameters than did single ones. Hence, nemastrol and humisun in concomitant with bio-arc, sweet basil callus, and oxamyl (half recommended dose) induced significant (P ≤ 0.05) and maximum improvement in total plant fresh weight and shoot dry weight. Similar trend was also noticed in root diameter and number of leaves of sugar beet infected with Meloidogyne spp. Additionally, the greatest suppression in nematodes’ population (95.7%), root galling (83.0%), and number of egg masses (100%) was also sustained at the soil amended with nemastrol + humisun + bio-arc + sweet basil callus + oxamyl since incorporation of such organic materials into soil might enhance B. megaterium activity that initiates antibiotics towards nematode population. However, single application of dried sweet basil callus showed better performance than did the dried leaves in terms of female fecundity and total nematodes’ population. Thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) indicated the presence of higher content of triterpenoides that belong to three groups, i.e., lupane, ursane, and oleanane, in dried sweet basil callus compared to native dried leaves powder. Concomitant treatment with nemastrol + humisun + bio-arc + sweet basil callus + oxamyl exhihited significantly increased in sucrose (17.1%), total sugar solids (21.8%), and purity (79.0%). The activities of both peroxidase (PO) and polyphenol oxidase (PPO) showed detectable fluctuations at the end of the experiment compared to untreated plants.

Background

Root-knot nematodes (RKNs) Meloidogyne spp. are worldwide plant pathogens playing a detectable role in limiting the productivity of economic agriculture crops in temperate regions. RKN, Meloidogyne incognita (Kofoid & White) Chitwood, is among the most important nematode species in sugar beet fields (Korayem 2006) causing damage to the epidermis, cortex, and stele regions which leads to giant cells and galls formation on fibrous and lateral roots that affect water and nutrient absorption (El-Nagdi and Abd El Fattah 2011). Biological control and other eco-friendly disease control measures have gained increasing interest among researchers after the environmental restrictions on nematicidal use for controlling plant parasitic nematodes. Plant growth rhizobacterium (PGPR) that belongs to Bacillus spp. is being exploited commercially for plant protection to induce systemic resistance against various pests and pathogens (Mostafa et al. 2014). However, several plant species have been demonstrated to have nematicidal activity against plant parasitic nematodes (Salim et al. 2016; El-Deriny 2016; Khairy 2016). Callus tissues are among plant parts that have been evaluated for their nematicidal effects and introduced to pharmacological research tests (Rateb et al. 2007). Many researchers have investigated the antifungal and antimicrobial activities of certain plant callus extracts (Shariff et al. 2006), but little attention has been given to their use in nematode management (Rocha et al. 2004; Osman et al. 2008; Nour El Deen 2008; Nour El-Deen and Darwish 2011). Several studies demonstrated that callus cultures of sweet basil accumulate various phenolic compounds with antioxidant activity (Makri and Kintzios 2008).

The present work was carried out in order to study the impact of certain bio-agents as resistance inducers singly or concomitantly on Meloidogyne spp. infecting sugar beet under field conditions.

Materials and methods

Tested bio-agents

• A) Bio-arc

  A native commercial formulation of phosphorus soluble bacterium, Bacillus megaterium (25 × 10⁶ cfu/g) at 2.5 g/L of distilled water

• B) Nemastrol

  A native commercial formulation of active ingredients containing glycosynolates (12%), chitinase (12 × 10⁵ IU), cytokinins (200 ppm), flavonoids (5%) and β 1–3, Glucanase (2 × 10⁵ IU) at the rate of 5 L/feddan

• C) Humisun

  A native commercial formulation of humic acid

• D) Sweet basil Ocimum basilicum callus

  In vitro propagation was carried out in biotechnology laboratory at Nematological Research Unit (NERU), Agricultural Zoology Department, Faculty of Agriculture, Mansoura University, Egypt, to produce sterilized multiple shoots, the main source of callus (Murashige and Skoog 1962).

• E) Sweet basil Ocimum basilicum leaf

  Native fresh leaves of O. basilicum were sun dried and powdered.

Experimental site

A micro-plot field experiment was carried out in a loamy soil (a mixture of coarse sand (2.9%), fine sand (20.7%), silt (39.8%), and clay (36.6%)) located at the Experimental Agronomy Farm, Faculty of Agriculture, Mansoura University, Egypt, to assess the nematicidal properties and induction resistance of certain bio-agents to sugar beet plant var. Negma. The plots were naturally infested with Meloidogyne spp. (308J₂/250 g), Rotylenchulus reniformis (453 immature females/250 g), and other nematode genera, i.e., Tylenchorhynchus, Helicotylenchus, Aphelenchus, and Xiphinema.

Agricultural practices

Inorganic fertilizers, i.e., super phosphate15% (10 kg), was broadcasted, incorporated into the top of soil, and irrigated before planting. Urea as nitrogen fertilizer (30 kg) and potassium sulfate (10 kg) were introduced 1 month after planting. Urea was reapplied after 2 weeks.

Design and micro-plot field layout

A field experiment, an area of 140 m², with a randomized complete block design (RCBD) and replicated three times was practiced. Each block included untreated control and 12 treated plots. A plot consisted of two rows, 60 cm wide and 5 m long. Plots were then planted with seeds of sugar beet var. Negma (three to four seeds/hill). Plants were thinned to one seedling/hill after 30 days of germination. One week after urea fertilization, all treatments were introduced. Oxamyl was applied at a rate of 0.3 g/plant in a single application and at half dose (0.15 g/plant) in concomitant applications. Plots were treated by the bio-control agents, i.e., bio-arc (a commercial product of Bacillus megaterium (20 ml/plant)); nemastrol (a mixture of active ingredients at 0.25 ml/plant) was applied two times at 1-week interval; humisun (a commercial product of humic acid) was applied at the rate of 100 ml/plant and powdered dried sweet basil callus (O. basilicum) as well as native leaves were applied three times at 1-week interval at the rate of 0.1 g/plant and incorporated into soil. Plants were harvested 6 months after planting, and roots were washed free from adhering soil. Data dealing with number of leaves, fresh shoot and root weight, dry shoot weight, shoot and root length, and root diameter were recorded. From each plot, a composite soil (250 g) was processed for nematode extraction by sieving and modified Baermann technique (Goodey 1957). At each treatment, root hairs (1 g) were stained in 0.01 acid fuchsine lactic acid (Bybd et al. 1983) and examined for the developmental stages, females, galls, and egg masses under a stereomicroscope.

Chemical analysis

One gram of dry weight of roots from each treatment was subjected to chemical analysis in order to evaluate total sugar solids (TSS), sucrose, and sugar purity.

Enzyme activity

Peroxidase (PO) and polyphenol oxidase (PPO) activities were determined in dried root tissues (0.5 g), according to the methods of Amako et al. (1994) and Coseteng and Lee (1987), respectively.

Determination of rosmarinic acid (RA) and terpenoid compounds

The presence of rosmarinic acid and terpenoids in methanolic extract of dried callus and powdered leaves of sweet basil was evaluated, using thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) (Kintzios et al. 2003). Authentic rosmarinic acid was used as standard. Extractions and measurements of both rosmarinic acid and terpenoides were carried out at the Faculty of Pharmacy, Mansoura University, Egypt.

Data analysis

Statistically, the obtained data were subjected to analysis of variance (ANOVA) (Gomez and Gomez 1984), followed by Duncan’s multiple range tests to compare means (Duncan 1955).

Results and discussion

Application of bio-arc (B. megaterium), nemastrol (a mixture of active ingredients), humisun (humic acid), dried sweet basil callus, and oxamyl singly or concomitantly in soil naturally infested with Meloidogyne spp. revealed that integrated of two or more components gave better results in sugar beet growth parameters than did single ones (Table 1). Single application of humisun (98%) and B. megaterium (58%) performed the best in ameliorating total plant fresh weight. Whilst growth parameters were significantly promoted (P ≤ 0.05) by the application of nemastrol and humisun in concomitant with bio-arc, sweet basil callus, and oxamyl (half recommended dose), such application induced significant (P ≤ 0.05) and maximum improvement in shoot (47.7%) and root (18.1%) lengths. Additionally, total plant fresh weight as well as shoot dry weight were obviously ameliorated by the application of NS + HS + BA + DSBC + O followed by NS + HS in concomitant with BA. Similar trend was also noticed with root diameter and number of leaves of sugar-beet infected with Meloidogyne spp. These findings supported the reports that humic acid at 0.04% not only offers significant nematode control but also improves growth of banana infected with M. incognita (Seenivasan and Senthilnathan 2017). Also humic acid treatments improved the yield of grape infected with M. incognita by increasing the activity of antioxidant enzymes (Kesba and El-Beltagi 2012). The rhizobacteria that belong to Bacillus viz. B. subtilis, B. megaterium, and B. pumilus have shown nematicidal activity against M. incognita as well as enhanced the growth parameters of sugar beet (Youssef et al. 2017). B. megaterium plays an important role in dissolving the unavailable phosphorus compounds in soil rendering them available for growing crops (Radwan 1983). However, oxamyl (standard nematicide) exhibited moderate increment in shoot weight (68.4%), total plant fresh weight (40.0%), root diameter (11.8%), and number of leaves (35.3%) of sugar beet (Table 1).

Table 1 Impact of certain bio-agents singly and concomitantly on plant growth parameters of sugar beet var. Negma infected with Meloidogyne spp. under field conditions

All tested materials showed antagonistic potential against Meloidogyne spp. infecting sugar beet. The integration of B. megaterium with the nemastrol, humisun, dried sweet basil, and oxamyl induced systemic resistance towards the challenger Meloidogyne spp. in sugar beet. Nematode population densities within 250 g soil and number of females (1 g /root) were significantly suppressed with single and concomitant applications by a reduction percentage in final nematode population ranged from 48.2 to 95.7% (Table 2). The greatest reduction in nematode population was sustained by the application of NS + HS + BA + DSBC + O (95.7%). Root galling (83.0%) and egg masses number (100.0%) were significantly suppressed for such treatment. Chitinase plays an important role in hydrolyzing chitin, the structure component in egg shell, and thus reducing nematode multiplication. Therefore, nemastrol (chitinase12 × 10⁵ IU) could be speculated to have a defense role during infection causing severe adverse effects on crucial biological processes of Meloidogyne spp. Results of this study support the findings of Mostafa et al. (2014) and El Deriny (2016) in respect of microbial activity, i.e., B. megaterium in the soil is enhanced on incorporation of organic matter that initiated antibiosis towards the nematode activity. Padgham and Sikora (2007) reported that B. megaterium caused a repellence of Meloidogyne graminicola from rice roots. Production of repellent substances or modification of the plant’s exudates by the antagonistic bacteria were suggested as mechanisms for this effect (Sikora et al. 2007).

Table 2 Impact of certain bio-agents singly and concomitantly on reproduction of Meloidogyne spp. infecting sugar beet var. Negma under field conditions

Sweet basil, O. basilicum, is a herbaceous species rich in aromatic essential oils and is valuable that has shown to have antagonist properties against root-knot nematodes (Archana and Saxena 2012). Herein, sweet basil callus (53.5%) and dried leaf powder (48.2%) exhibited the least reduction in nematodes’ population. However, dried sweet basil callus showed better performance than did dried leaves in terms of female fecundity. Thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) indicated the presence of high content of triterpenoides that belong to the three groups, i.e., lupane, ursane, and oleanane, in dried O. basilicum callus compared to native dried leaves powder. Meanwhile, O. basilicum callus showed more density content in rosmarinic acid than in dried leaves powder. Rosmarinic acid has a number of interesting biological activities, i.e., antiviral, antibacterial, anti-inflammatory, antinematode (Caboni et al. 2013), and antioxidant. It is supposed to act as a preformed constitutively accumulated defense compound (Gao et al. 2005).

On the other hand, untreated sugar beet plants showed a decrease in size of storage roots and malformation appearance (forked roots) with percentage value amounted to 26.7 (Table 2). Less branched roots were noticed with the introduction of sweet basil callus, sweet basil dried leaf powder, and oxamyl. However, sugar beet plants showed healthy storage root with NS + HS + BA + DSBC +O and NS + HS + BA as well. Technological characters in terms of sucrose (17.1%), TSS (21.8%), and purity (79.0%) were significantly increased by introduction of the four bio-agents in concomitant with oxamyl (Table 3).

Table 3 Impact of certain bio-agents on technological characters as well as peroxidase (PO) and polyphenol oxidase (PPO) activities in roots of sugar beet var. Negma infected with Meloidogyne spp. under field conditions

Increased activity of defense-related enzymes, i.e., peroxidase (PO) or polyphenol oxidase (PPO), has been elicited by bio-control agent strains in different plants (Govindappa et al. 2010). PO and PPO are thought to reinforce cell walls (lignification and suberization) at the border of infection and further limit spread of pathogens (Passardi et al. 2004). Previous studies reported that application of bio-arc + nemastrol under greenhouse conditions increased the activities of related enzymes, i.e., PO and PPO, in roots of sugar beet infected with M. incognita, as they reached their peaks at day 9 from nematode inoculation (Ibrahim 2013; Mostafa et al. 2014). In current investigation, the activities of PO and PPO were evaluated at the end of the experiment and showed detectable fluctuations among all treatments. The greatest activities of PPO and PO were recorded in control plants. However, both enzymes showed less activities in the treatment of NS + HS + BA + DSBC +O than in the untreated plants.

Conclusions

The use of screened organic materials, i.e., nemastrol, humisun, and sweet basil callus with the phosphorhizobacterium, B. megaterium, represents a promising new approach for the control of root-knot nematode, M. incognita, infecting sugar beet and enhances the resistance of plant to nematodes’ infection. Moreover, the importance of using natural resources instead of synthetic antioxidants has risen globally. Therefore, attempts to increase active compounds in ornamental plants, i.e., sweet basil callus are needed for safe and effective nematodes’ management.