Influence of hydrogen cyanide-producing rhizobacteria in controlling the crown gall and root-knot nematode, Meloidogyne incognita

Abstract

Out of 39 isolates of rhizobacteria, recovered from economic plants grown in 8 locations in Egypt, 6 isolates were able to produce Hydrogen Cyanide (HCN). 16S rRNA sequence analysis identified these isolates as: Pseudomonas japonica strain NBRC 103040, Bacillus megaterium strain CtST3.5, Pseudomonas sp. strain Gamma-81, P. tolaasii strain ATCC 33618, P. chlororaphis strain Lzh-T5, and P. mosselii strain CV25. These HCN producers were able to inhibit growth of Agrobacterium tumefaciens and affect viability of Meloidogyne incognita juveniles in vitro. The isolates of P. japonica and Pseudomonas sp. Gamma-81 prevented the gall formation on tomato plants by A. tumefaciens, regardless of the presence of M. incognita. The isolates of B. megaterium, P. chlororaphis, P. tolaasii, and P. mosselii decreased the weight and number of galls produced by A. tumefaciens in the presence or absence of M. incognita. The 6 HCN producers decreased the population of M. incognita and the number of nematode galls than the positive control, when used against M. incognita. A similar effect was achieved against mixed infections with M. incognita and A. tumefaciens. The HCN-producing rhizobacteria, in the presence of A. tumefaciens and/or M. incognita, caused obvious increment in all growth parameters of tomato than the negative control and healthy plants. The only exception was found in case of Pseudomonas sp. Gamma-81 against M. incognita and against mixed infection, where growth parameters of tomato were decreased. Although the isolates were naturally isolated from the rhizosphere of economic plants, it must be cautiously considered since the isolate identified as P. japonica has been reported as a human pathogen. Also, P. tolaasii was reported causing a bacterial blotch on cultivated mushrooms under certain environmental conditions. Further investigations are needed.

Background

Crown gall disease caused by Agrobacterium tumefaciens is a major bacterial disease in nurseries and orchards and is being considered one of the most important disorders that causes high losses in nurseries (Pulawska 2010). Biological control of crown gall is more effective in controlling the disease rather than chemical control (Tolba and Soliman 2013). Root-knot nematodes, Meloidogyne spp., are among the most important plant fauna that limit the productivity of many economic crops. The search for biological means for controlling root-knot nematodes (RKNs) is important because of inability of plants to resist most types of RKNs, and the chemical control is subject to debate due to the environmental hazards and human health damage (Mostafa et al. 2014). The interrelation between nematode densities and increasing of crown gall on the roots was reported for many crops references. Occurrence of wounds by nematodes allows incorporation of T DNA (the transferred DNA of the tumor-inducing (Ti) plasmid) of bacteria into the genome of plant cell and development of crown gall disease (Rubio-Cabetas et al. 2001).

Cyanide forms stable complexes with the essential elements (Cu²⁺, Fe²⁺, and Mn²⁺) for the protein function and therefore is considered a toxic substance to most living organisms. Hydrogen cyanide (HCN) is a volatile secondary metabolite that is synthesized by many rhizobacteria and has a powerful effect on many organisms. HCN inhibits the electron transport and disrupts the energy supply to the cell, which leads to death of living organisms. Many bacterial genera have the ability to produce HCN including species of Alcaligenes, Aeromonas, Bacillus, Pseudomonas, and Rhizobium (Alemu 2016).

The ability of some bacterial strains to control nematodes was attributed to their ability to produce HCN. Moreover, a direct influence of contact between HCN-producing bacteria and the nematode is essential in the effective control of target nematode (Siddiqui et al. 2006). HCN may affect plant establishment or inhibit development of plant disease, with great potential for controlling plant bacterial diseases (Lanteigne et al. 2012). Some studies have questioned of HCN based on the lack of correlation of the level of HCN produced by rhizobacteria with the biocontrol effects in vitro. These studies have suggested that HCN does not act directly in the process of biocontrol, but involved in geochemical processes in the substrate (e.g., chelation of metals) leading to an indirect increase in the nutrients availability for the rhizobacteria and their plant hosts (Rijavec and Lapanje 2016).

The purposes of this work were (1) to isolate and identify HCN-producing rhizobacteria from different locations in Egypt, screen the ability of these rhizobacteria in inhibiting A. tumefaciens, and evaluate their influence on Meloidogyne incognita in vitro; (2) to study the correlation of HCN levels produced by rhizobacteria with the biocontrol effects in vitro; and (3) to study the influence of these HCN-producing rhizobacteria on the growth of tomato plant, the crown gall disease, the root-knot nematode infection, and the mixed infection by these two pathogen in vivo.

Materials and methods

Sources of the pathogens

The crown gall bacterium, A. tumefaciens (virulent isolate), was previously isolated from peach and identified by Abd El-Rahman (2012). The pathogenicity test was re-performed to check virulence. In order to confirm the identification, the 16S rDNA analysis was used. The sequencing process was conducted at the Potato Brown Rot Project laboratories (Giza, Egypt), using forward primer (U968-f) 5′-AACGCGAAGAACCTTAC-3′ and reverse primer (L1401-r) 5′-CGGTGTGTACAAGACCC-3′ as described by Farag et al. (2017). The RKN, M. incognita inoculum, was obtained from Nematode Diseases Research Department, Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt.

Isolation of rhizobacteria

Rhizobacteria were isolated from rhizosphere of different crops from 8 locations in Egypt. Serial dilution plate count technique was followed, using soil adhering to the roots of screened plants as described by Kifle and Laing (2016). Nutrient glucose agar and King’s B agar media were used for the isolation of bacteria. The plates were incubated at 28 °C for 3 days. The separate colonies were selected and stored as pure cultures.

Screening hydrogen cyanide (HCN) produced by rhizobacterial isolates

Assessment of HCN produced by rhizobacterial isolates was carried out. Appropriate amount of each isolate was inoculated in 250-ml flask (3 flasks/isolate), containing King’s B broth medium (100 ml) amended with 4.4 g/l glycine. Non-inoculated flasks were used as a control. Sterile filter paper strip was dipped in picric acid solution (0.5% picric acid in 2% sodium carbonate) and was attached to the neck of the flask. The flask was plugged and sealed off with Parafilm. Incubation was made at (28 ± 2 °C) for 4 days, with shaking at 140 rpm. A change in color of the filter paper strips from yellow to light brown, brown, or brick red was recorded as weak (+), moderate (++), or strong (+++) reaction, respectively. No change in color was recorded as a negative (−) reaction (Abd El-Rahman and Shaheen 2016).

Identification of selected isolates of rhizobacteria

Identification of selected rhizobacteria was made, using 16S rRNA analysis at Sigma Scientific Services Co., Giza, Egypt, as described by Abd El-Ghany et al. (2017). DNA extraction was made, using of GenJet™ genomic DNA purification Kit (ThermoK0721), according to the manufacturer’s protocol by gram-negative bacteria genomic DNA purification protocol. PCR was made, using Maxima Hot Start PCR Master Mix (Thermo K 1051), according to the manufacturer’s protocol by using forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse primer 5′-GGTTACCTTGTTACGACTT-3′ (Eden et al. 1991). The PCR products were purified using GeneJet™ PCR purification Kit (Thermo K0701). Finally, the PCR products were sequenced, using ABI 3730 × 1 DNA sequencer, using forward and reverse primers. Obtained sequences were compared to those available in the database GeneBank network services at http://blast.ncbi.nlm.nih.gov/Blast.cgi for significant alignments.

Quantitative assay of hydrogen cyanide (HCN) production

Suspension (3 ml of 10⁶ cfu/ml) of each bacterial isolate was inoculated in (250 ml) flask containing King’s B broth medium (100 ml) amended with glycine (4.4 g/l). Three replicates were used for each tested isolate. Non-inoculated flasks were used as control. Strips of filter paper (10 × 0.5 cm) were soaked in alkaline picrate solution and kept hanging to the neck of the flask (one strip for each flask). The flasks was plugged and sealed off with Para film. Three replicates were used for each isolate. After incubation at 28 ± 2 °C for 4 days (with shaking at 140 rpm), the color of sodium picrate strips was changed to a reddish compound in proportion to the amount of HCN evolved. The color was eluted by placing the changed filter paper in a test tube containing 10 ml of distilled water, and its absorbance was read by the UV/Visible spectrophotometer (MODEL:2000 UV – UNICO INSTRUMENTS CO., LTD,USA) at 625 nm. Distilled water was used to adjust the zero absorbance reading at 625 nm before reading (Reetha et al. 2014).

Preparation of A. tumefaciens and rhizobacteria inoculum

Each bacterial isolate was grown in King’s B agar medium plates for 48 h at 28 °C, and then, bacterial growth was harvested into a saline solution (0.85% NaCl) to prepare bacterial suspension that was adjusted to 10⁸ cfu/ ml.

Preparation of M. incognita inoculum

Fresh second-stage juveniles (J₂) of M. incognita were obtained from pure culture maintained on coleus (Coleus blumei) roots. Roots were incubated for 5–7 days in a modified Baermann method for hatching at room temperature (Ibrahim et al. 2013).

In vitro antagonistic determination of rhizobacteria against gall-forming bacteria and second-stage juveniles

Certain rhizobacterial isolates were screened for their ability to inhibit the growth of A. tumefaciens on KBA medium plates. A loopful of the tested isolate (24 h old culture) was placed at the center of inoculated plate with A. tumefaciens, using 3 ml of 10⁸ cfu/ml suspension for flask containing 250 ml medium. Three replicates were used for each tested isolate. The plates were incubated at 28 °C for 48 h, and the inhibition zone diameter was measured to the nearest millimeter (Abd El-Rahman and Shaheen 2016).

Detrimental effect of bacterial isolates against (J₂) of M. incognita was evaluated. One milliliter (10⁸cfu/ml) of bacterial isolates was added to 4 ml of M. incognita suspension containing 100 juveniles (J₂) in a Petri dish (6 cm diameter). One milliliter of saline solution (0.85% NaCl) instead of bacterial suspension was used as control. Three replicates from each treatment were made. All dishes were sealed off with Parafilm and kept at the room temperature (30 °C). The dishes were examined under a microscope after 24, 48, and 72 h and then left for 7 days and checked for the last time. The number of surviving and dead larvae was counted. Percentage mortality due to the effect of bacterial isolate was calculated as follows:

Mortality % = [(number of live nematode juveniles in control − number of live nematode juveniles in treatment)/number of live nematode juveniles in control] × 100 (Abdel-Salam et al. 2018).

In vivo experiment

The effect of selected isolates of rhizobacteria on growth of tomato plant, crown gall disease, the RKN, and the mixed infection by these 2 diseases was tested in a greenhouse (22 ± 3 °C). Each pot (20 cm in diameter) containing 800 g clean soil (clay to sand to compost at 2:1:1 ratio) was planted by one of 3 weeks old tomato (cv. Super Strain B) seedlings. The first group of pots was allocated to study the effect of rhizobacteria on crown gall disease, the second one for the effect of rhizobacteria on RKN, and the third was to test the effect of rhizobacteria on the mixed infection with crown gall bacteria and root-knot nematode. Pots were arranged in a completely randomized design and treated as needed experimentally. Five replicates per treatment were used. All treatments were run 10 days after planting. Five pots were left free of nematode, A. tumefaciens, and rhizobacteria as un-inoculated (healthy plant) treatment for the 3 experiment groups. Five pots pricked at the crown region of tomato plants were used as negative control for the first and third experiment groups.

For the first group, the seedlings were pricked at the crown region of tomato plants, and the soil was drenched with suspension (10⁸ cfu/ml) of bioagent (100 ml/pot) along with suspension (10⁸ cfu/ ml) of A. tumefaciens isolate (100 ml/pot) together at the same time. Five inoculated pots with A. tumefaciens were served as a positive control. For the second group, the soil was drenched by the suspension (10⁸ cfu/ml) of bioagent at the rate of 100 ml/pot (five pots treated with oxamyl 0.3 ml ̸pot was used for comparison), and after 48 h, inoculation was made with 2000 s-stage juveniles/plant of M. incognita. Five inoculated pots with M. incognita were served as a positive control. The same treatments in the first group were prepared as a third trial, and after 48 h, the seedlings were inoculated by 2000 of the second-stage juveniles/plant of RKN. Five inoculated pots with A. tumefaciens and M. incognita served as a positive control.

Pathological determination

Determination for A. tumefaciens and M. incognita syndromes were recorded after 45 days of inoculation. Weight (g) and number of galls of A. tumefaciens were recorded, in the first and the third groups. Percentage of decreasing in crown gall disease (PDD) was calculated from the weight and number of galls as follows: PDD = [(C − T (/ C] × 100 where C = weight or number of galls in control and T = weight or number of galls in treatment.

Nematode population in soil (250 g), number of developmental stages and females in roots, number of galls, and number of egg masses of M. incognita were recorded in the second and the third groups of treatments. The final population (Pf) of nematode was calculated as follows: nematode population in soil + no. of developmental stages + no. of females. Reproduction factor (RF) was calculated as follows: final population (Pf) in treatment/no. of treatment initial inoculum. The percentage of reductions in nematode population (red %) was calculated as follows: Red% = [(Pf_c − Pf_t)/Pf_c] × 100 where Pf_c = the final population in control and Pf_t = the final population (Pf) in treatment. Root gall index (RGI) or egg masses index (EI) was determined according to the scale given by Taylor and Sasser (1978) as follows: 0 = no galls or egg masses, 1 = 1–2 galls or egg masses, 2 = 3–10 galls or egg masses, 3 = 11–30 galls or egg masses, 4 = 31–100 galls or egg masses, and 5 = more than 100 galls or egg masses.

Plant growth parameters

Growth parameters (shoot length, shoot fresh weight, and root fresh weight) of tomato plants were recorded after 45 days of inoculation at all treatments. The dry weight of the whole plant is estimated after drying the vegetative growth and roots of plant in oven at 100 °F (37.8 °C) overnight.

Statistical analysis

Completely randomized design was used in all in vitro and in vivo pot experiments. Collected data were subjected to analysis of variance. Comparisons among treatment means were made, using Duncan multiple range test at 0.05 level of probability, using MINITAB software (version 16, MINITAB, Inc., State College, PA).

Results and discussion

Confirmation the identity of the crown gall bacterium Agrobacterium tumefaciens (virulent isolate)

Bacterial isolate, previously identified as A. tumefaciens on the basis of the traditional bacteriological methods, was further confirmed by 16S rDNA analysis. The results showed that A. tumefaciens (virulent isolate) showed (98.41%) similarity with A. tumefaciens strain IAM 12048 and A. tumefaciens strain NCPPB2437. Distance tree, using rRNA type strains/Bacteria and Archaea_16S_ribosomal RNA sequences database, is illustrated in (Fig. 1).

Fig. 1

Distance tree using rRNA type strains/bacteria and Archaea_16S_ribosomal RNA sequences database. lcl|Query _ 175837 refers to the A. tumefaciens (virulent isolate)

Ability of rhizobacteria to produce hydrogen cyanide (HCN)

Many bacterial genera had the ability to produce HCN embracing many species of Alcaligenes, Aeromonas, Bacillus, Pseudomonas, and Rhizobium (Alemu 2016). Thirty-nine isolates of rhizobacteria, recovered from different locations and different crops in Egypt, were tested for their ability to produce hydrogen cyanide (HCN). Only 6 isolates were able to produce HCN. The 6 HCN producing isolates were isolated from rhizosphere soil of guava plants grown in Behiera governorate (BR11 and BR13), peach plants grown in Giza governorate (GA21), potato plants grown in Ismailia governorate (SW1 and SW4), and corn plants grown in Beni-Seuf governorate (BW3). A strong (+++) HCN production was recorded by the isolate BR13 from guava; while a moderate (++) reaction was recorded by BR11, GA 21, and SW1 from guava, peach, and potato, respectively. A weak (+) reaction was recorded by BW3 and SW4 from corn and potato, respectively (Table 1).

Table 1 Source of rhizobacterial isolates and their tentative ability to produce HCN

16S rRNA analysis

Results of DNA sequence, obtained from 16S rRNA analysis of the 6 HCN isolates collected from different plant rhizosphere and locations in Egypt compared to DNA sequence available at the NCBI database, are shown in (Table 2). Comparison of the DNA sequences of SW1, BW3, SW4, BR 11, BR 13, and GA 21 with DNA sequence in the NCBI database identified these isolates as: Pseudomonas japonica strain NBRC 103040, Bacillus megaterium strain CtST3.5, Pseudomonas sp. strain Gamma-81, P. tolaasii strain ATCC 33618, P. chlororaphis strain Lzh-T5, and P. mosselii strain CV25 by percent of 97, 98, 100, 99, 99, and 99), respectively.

Table 2 Molecular identification of six plant rhizosphere isolates using 16S rRNA gene sequence

Quantitative assay of HCN production by bacterial isolates

Quantitative assay of HCN production by bacterial isolates showed that P. chlororaphis greatly produced HCN and recorded the maximum absorbance value of (0.047), followed by P. mosselii, P. tolaasii, and P. japonica, which recorded absorbance values of 0.027, 0.024, and 0.020), respectively. The lowest absorbance values (0.002 and 0.005) were recorded by B. megaterium and Pseudomonas sp. Gamma-81, respectively (Table 3).

Table 3 Quantitative assay of HCN produced by bacterial isolates at 625 nm wavelength

Efficacy of HCN-producing rhizobacteria on growth of A. tumefaciens and M. incognita in vitro

The results reported herein (Table 4) showed that the 6 isolates of HCN-producing rhizobacteria could inhibit the growth of A. tumefaciens in vitro. The inhibition zone diameter ranged from 13.7 to 49.7 mm. The maximum inhibition (49.7 mm) was recognized by the P. japonica isolate, followed by P. mosselii, Pseudomonas sp. strain Gamma-81., P. chlororaphis, P. tolaasii, and B. megaterium, which recorded 33.7, 30.7, 25.7, 14.3, and13.7 mm), respectively.

Table 4 Effect of six isolates of HCN- producing rhizobacteria on growth of Agrobacterium tumefaciens in vitro

Efficacy of 6 isolates of HCN-producing rhizobacteria on M. incognita under laboratory conditions was shown in (Table 5) as a percentage of mortality of 100 juveniles (J₂) of M. incognita after different exposure periods to bacterial suspension. The results showed that P. japonica, P. chlororaphis, and P. mosselii had influenced J₂ survival after 24 h incubation. The mortality rate of juveniles reached 24.3, 38.0, and 23.0%, respectively. By increasing the exposure time, all bacterial isolates affected the life of nematode juveniles. The rate of mortality of the juveniles ranged from 23.7 to 49.2% and from 32.3 to 68.6% after 48 and 72 h, respectively. After 7 day of incubation, the maximum mortality percent was achieved by P. chlororaphis isolate, recording 96.4 with significant differences with Pseudomonas sp. Gamma-81, P. mosselii, P. japonica, B. megaterium, and P. tolaasii, which recorded mortality percent of 63.9, 54.6, 51.8, 43.9, and 42.8, respectively.

Table 5 Effect of different exposure time with certain HCN-producing rhizobacteria on mortality rate of Meloidogyne incognita

In this regard, some studies have questioned the biocontrol effect of HCN based on the findings on the lack of correlation between the level of HCN produced by rhizobacteria and the antagonistic effects in vitro (Rijavec and Lapanje 2016). From these results, the correlation of the level of HCN, produced by rhizobacteria with the biocontrol effects in vitro, was more clearly in the case of M. incognita nematode than the case of A. tumefaciens bacteria, although this correlation was not a complete correlation relationship in the both cases.

Effect of HCN-producing rhizobacteria on incidence of crown gall on tomato plants

Effect of 6 isolates of HCN-producing rhizobacteria on incidence of crown gall caused by A. tumefaciens on tomato plants in the absence or presence of M. incognita after 45 days of inoculation was shown in (Table 6). Results proved that the isolates of P. japonica and Pseudomonas sp. Gamma-81 prevented completely gall formation on tomato plants in both cases. In the absence of M. incognita, the isolates of B. megaterium, P. chlororaphis, P. tolaasii, and P. mosselii decreased the weight of galls caused by A. tumefaciens on tomato plants by 92.1, 67.0, 60.8, and 49.9%, respectively. Also, a pronounced decrement in the number of galls by 93.3, 52.0, 56.0, and 48.0% was recognized, respectively. In the presence of M. incognita, the isolates of B. megaterium, P. chlororaphis, P. tolaasii, and P. mosselii decreased the gall weight produced by A. tumefaciens on tomato plants by 80.2, 81.7, 64.8, and 53.0% and decreased the number of galls by 81.7, 73.9, 62.5, and 57.0%, respectively. Although the isolate of B. megaterium showed a high ability to control crown gall disease in the absence of M. incognita, however, this capability was decreased significantly in the presence of M. incognita by 11.9% for gall weight and by 11.6% for number of galls. Meanwhile, though the isolates of P. tolaasii, P. chlororaphis, and P. mosselii showed a moderate ability to control crown gall in the absence of M. incognita, the capability increased in the presence of M. incognita by 4.0, 14.0, and 3.1% for gall weight and by 6.5, 21.9, and 9.0% for the number of galls, respectively. This is despite the fact that P. chlororaphis and P. mosselii produced HCN and inhibited the growth of A. tumefaciens better than Pseudomonas sp. Gamma-81 in vitro. These results denoted that HCN can play a role in controlling the crown gall disease, in addition to possible other factors produced by antagonistic bacteria that are more potent in controlling the disease. There are many mechanisms that are used by Pseudomonas spp. to control plant diseases such as production of some secondary metabolites (hydrogen cyanide, Fe-chelating siderophores, and antibiotics), induction of plant defense responses and competition for nutrients (Lukkani and Reddy 2014).

Table 6 Effect of six isolates of HCN-producing rhizobacteria on incidence of crown gall disease on tomato plants in the absence or presence of Meloidogyne incognita after 45 days of inoculation

Effect of HCN-producing rhizobacteria on incidence of root-knot nematode on tomato plants

The potential of some bacterial strains to control nematodes is related to their ability to produce HCN. Moreover, a direct contact between HCN-producing bacteria and the nematode is essential in the effective control of nematode (Siddiqui et al. 2006). Data in Table 7 showed that HCN-producing rhizobacteria decreased the final population of M. incognita and the number of nematode galls than the positive control, when used against M. incognita and mixed infection with M. incognita and A. tumefaciens. The lowest final population count of nematodes (411.0 and 342.4) was recorded by P. mosselii, when used against M. incognita and mixed infection, respectively. The M. incognita and mixed infection (positive controls) recorded final population counts of 5574.0 and 4560.4, respectively. The nematicidal oxamyl recorded a final population count of 288.0, when used as a comparison treatment against M. incognita.

Table 7 Effect of six isolates of HCN-producing rhizobacteria on incidence of root-knot nematode on tomato plants in the absence or the presence of A. tumefaciens after 45 days of inoculation

The lowest population counts of nematode in soil (396.0 and 324.0) were recorded by P. mosselii, when used against M. incognita and mixed infection, respectively. The M. incognita and mixed infection (positive controls) recorded population counts in soil of 5412.0 and 4456.0, respectively. Oxamyl recorded a population count in soil of 244.0 in soil, when used as a comparison treatment against M. incognita.

The number of nematode galls decreased by using HCN-producing rhizobacteria against M. incognita and mixed infection than the positive control. The lowest numbers of galls (11.2 and 11.0) were recorded by P. japonica and P. mosselii, when used against M. incognita and mixed infection, respectively. The M. incognita and mixed infection (positive controls) recorded a number of galls of 63.8 and 53.0, respectively. Oxamyl recorded a number of galls (21.6), when used as a comparison treatment against M. incognita.

These results are remarkably consistent with the results obtained in the laboratory, where a quantitative assay of producing HCN by tested bacterial isolates showed that P. japonica, P. tolaasii, P. chlororaphis, and P. mosselii strongly produced HCN compared to B. megaterium and Pseudomonas sp. Gamma-81. Only P. japonica, P. chlororaphisi, and P. mosselii isolates affected the life of juveniles (J₂) of M. incognita after 24 h of incubation. This conclusion is clear in spite of the observation that isolate of P. japonica did not give good results that match with laboratory experiments.

Effect of HCN-producing rhizobacteria on growth parameters of tomato plants

The 6 isolates of HCN-producing rhizobacteria in the presence of A. tumefaciens and/or M. incognita increased growth parameters of tomato (shoot and root length, shoot and root fresh weight, total fresh weight of plant, and total dry weight of plant) than the negative control and healthy plants (Table 8). The only exception was found in case of Pseudomonas sp. Gamma-81, when used against M. incognita and the mixed infection (A. tumefaciens and M. incognita), where growth parameters of tomato were decreased. On the other hand, A. tumefaciens (positive control treatment) increased all growth parameters than the negative control and healthy plants, while M. incognita (positive control treatment) decreased all growth parameters than the healthy plants.

Table 8 Effect of HCN-producing rhizobacteria on growth parameters of tomato plants infected with of Agrobacterium tumefaciens and/or Meloidogyne incognita after 45 days of inoculation

When the tested HCN-producing rhizobacteria were used against A. tumefaciens, all isolates increased shoot weight, root length, total fresh weight, and total dry weight of tomato plants than the positive control treatment (A. tumefaciens). The only exception was recognized in case of root length and total fresh weight in P. japonica isolate treatment. The isolate of P. japonica recorded root length and total fresh weight of 28.2 cm and 49.3 g compared to A. tumefaciens (positive control treatment) that recorded 29.7 cm and 52.9 g, respectively. Also, P. japonica, B. megaterium, and P. chlororaphis increased shoot length compared to the positive control treatment (A. tumefaciens) and recorded 58.4, 56.3, and 61.8 cm, respectively, while Pseudomonas sp. Gamma-81, P. tolaasii, and P. mosselii decreased shoot length than the positive control treatment (A. tumefaciens) and recorded 52.5, 51.7, and 54.3 cm, respectively. The positive control treatment (A. tumefaciens) recorded shoot length of 55.0 cm. Meanwhile, root weight of tomato plants was increased by using P. mosselii against A. tumefaciens than the positive control treatment (A. tumefaciens) and recorded 18.0 g. At the same time, root weight of tomato plants was decreased by using P. japonica, B. megaterium, Pseudomonas sp. Gamma-81, P. tolaasii, and P. chlororaphis, which recorded 8.3, 12.6, 12.9, 14.0, and 11.0 g, respectively. The positive control treatment (A. tumefaciens) recorded root weight of 14.8 g.

Meanwhile, the tested HCN-producing rhizobacteria against M. incognita increased all growth parameters of tomato plants than the positive control treatment (M. incognita). The only exception was found in case of Pseudomonas sp. Gamma-81 that decreased all growth parameters of tomato plants.

HCN-producing rhizobacteria gave various results when used against mixed infection with A. tumefaciens and M. incognita. The isolates of P. japonica and P. tolaasii increased all plant growth parameters of tomato than the positive control treatment (A. tumefaciens and M. incognita), the only exception was found in case of root length. The isolate of B. megaterium increased all tomato plant growth parameters than the positive control treatment (A. tumefaciens and M. incognita), the only exception was found in case of shoot weight and total fresh weight. Also, P. mosselii increased all tomato plant growth parameters than positive control treatment (A. tumefaciens and M. incognita), except the root length and root weight. Meanwhile, Pseudomonas sp. Gamma-81 decreased all tomato plant growth parameters than the positive control treatment. Also, P. chlororaphis decreased shoot weight, root length, root weight and total fresh weight and increased shoot length and total dry weight compared to the positive control treatment.

In addition to its ability to promote the growth of plants indirectly by inhibiting the growth of pathogens, it is well established that some Pseudomonas spp. can produce substances that promote the growth of plants directly as indole-3-acetic acid (IAA), siderophores that chelate iron, and phosphatases that solubilize phosphorus (Rai et al. 2017). Moreover, Bacillus spp. beside its ability to act as a biological agent by producing HCN and antibiotics, they also produce phytohormones like IAA and GA, increases uptake of nutrients like phosphate and iron by siderophore production, produces ammonia, protects cell from oxidative damage by producing catalase enzyme and exhibited tolerance against heavy metal and salinity (Rayavarapu and Padmavathi 2016). More interesting, it has been recently found that A. tumefaciens isolate, isolated from nodules of plants growing in zinc-lead mine tailings, showed high metal resistance and enhanced the growth of plants in a metal-contaminated environment (Hao et al. 2012). On the other hand, M. incognita decreased the plants growth, causing loss in plant parameters that may be directly correlated with the multiplication of the nematodes (Anver and Alam 1989). The results herein showed that A. tumefaciens caused an increment in all plant parameters compared to the negative control and healthy plants, while M. incognita decreased all plant parameters than the healthy plants.

Finally, although all obtained isolates were recovered locally from rhizosphere soils of some plants and had a role in biocontrol of A. tumefaciens and M. incognita, but P. japonica and P. tolaasii cannot be used in this field, until being sure that these strains are not pathogenic to humans and animals. In this regard, it has been reported that P. japonica is a novel species in the sub-cluster of P. putida group, isolated from an activated sludge and more recently reported as a novel cause of skin bacteremia and soft tissue infection (Coomes et al. 2018). P. tolaasii is a species of gram-negative bacteria causing bacterial blotch on cultivated mushrooms (Agaricus bisporus) under some environmental conditions (Soler-Rivas et al. 1999). Thus, further field trials and pilot experimentation are suggested.

Conclusion

Six isolates of HCN-producing rhizobacteria were recovered locally from rhizosphere soils of some plants in Egypt. These HCN-producing rhizobacteria were able to inhibit growth of Agrobacterium tumefaciens and affect viability of Meloidogyne incognita juveniles in vitro. The isolates decreased the weight and number of galls produced by A. tumefaciens on tomato plants, regardless to the presence of M. incognita. The 6 HCN producers decreased the population of M. incognita and the number of nematode galls produced on tomato when used against M. incognita, and a similar effect was achieved against mixed infection with M. incognita and A. tumefaciens. The HCN-producing rhizobacteria in the presence of A. tumefaciens and/or M. incognita caused obvious increment in all growth parameters of tomato plant. Further investigations are still needed.