Background

Broomrape is a parasitic weed widespread in many parts of the world (Brhane et al. 2016) as well as in Egypt (Abdel-Kader and El-Mougy 2009; Ghannam et al. 2012), where it is a major factor limiting faba bean production (Vicia faba). Broomrape attacks a wide range of plant species such as carrot, celery, eggplant, faba bean, potato, peppers, peas and tomato. By chemicals released from host roots, the broomrape seeds could germinate, and their seedling roots attack the host plant roots to obtain water and nutrients and remain underground until flowering. Broomrape seeds can be spread by footwear, livestock, machinery, vehicles and flooding (Punia 2014). No single method was effective for economically managing Orobanche spp. Therefore, several alternative methods viz. preventive, physical, chemical, agronomic, biological, crop resistance and integrated management were applied for controlling the Orobanche. Therefore, we need a single cheap method to be effectively, economically and completely controlling this parasite (Habimana et al. 2014; Punia 2014).

The plant growth-promoting bacteria (PGPB) can play a role in protecting plants against Orobanche infection. Pseudomonas fluorescens showed a high bio-control activity against Orobanche crenata and Orobanche foetida and positively enhanced the plant growth parameters of faba bean (Zermane et al. 2007). Pseudomonas marginalis reduced the incidence of O. crenata and improved faba bean (Zermane et al. 2007). Bacillus circulans, Bacillus megatherium var. phosphaticum (BMP) and the combination of BMP plus Rhizobium leguminosarum bv. viceae significantly reduced the germination of O. crenata and increased the dry weight of faba bean (Elabaied et al. 2018).

Application of Trichoderma harzianum and Trichoderma viride, as soil treatment, followed by spray with glyphosate significantly reduced the broomrape infection and increased the yields of peas, faba bean and tomatoes in the fields, compared with each fungus treatment (Abdel-Kader and El-Mougy 2007, 2009).These treatments may be effective, applicable and cost-effective for controlling Orobanche. Several fungi also viz. Chaetomium sp., Fusarium oxysoprum, Fusarium solani, Rhizoctonia solani, Sclerotium rolfsii and T. harzianum could attack the living tissues of Orobanche ramose segments causing black lesions, soft rots and complete deterioration within 7 days (Nawar and Sahab 2011). Application of T. viride and T. hamatum reduced the number of Orobanche shoots (Nawar and Sahab 2011). Trichoderma harzanium, when applied single or in combination with Rhizobium legimunosarum, significantly reduced the disease severity of O. crenata and improved the seeds and biomass yield of faba bean varieties (Brhane et al. 2016). F. oxysporum, as conidial suspension, significantly decreased the germination and tubercles of Orobanche, where the microconidia and chlamydospore formulations greatly diminished the emerged shoots and the germinated seeds of Orobanche (Alla et al. 2008). Babalola (2010) found that infested tomato roots with Fusarium compactum mycelia plus pectinase treatment (20 U ml− 1) had over 50% tubercles dead after 1 week of treatment, while mycelia with mycelia plus cellulose treatment (20 U ml− 1) had above 60% mortality.

This work aimed to investigate the application of plant growth-promoting bacteria, i.e. P. fluorescens, Bacillus subtilis and Bacillus pumilus and Trichoderma spp., i.e. T. harzianum, T. viride and T. vierns for controlling O. crenata infestation and their effects on the growth parameters of faba bean under greenhouse conditions.

Materials and methods

Bio-control agents

The plant growth-promoting bacteria, i.e. P. fluorescens, B. subtilis and B. pumilus as well as T. harzianum, T. viride and T. vierns were obtained from Plant Pathology Department, National Research Centre, Egypt.

Herbicide

The herbicide Glyphosate (Glialka 48% WSC) was applied at recommenced dose (225 cm3 per hectare) as the control.

Preparation of bio-control agent’s inocula

For preparation of Trichoderma spp. propagules (colony-forming unit, CFU), the pure culture of each T.harzianum, T. viride and T. vierns fungal species was maintained on potato dextrose agar in Petri plates at 30 ± 2 °C. For mass production, 1-l conical flasks containing 500 ml of malt extract medium (MEM; 30 g malt extract, 5 g peptone in 1000 ml distilled water) were autoclaved. Then, each flask was separately inoculated with 1-cm-diameter disc of each Trichoderma sp. The flasks were inoculated at 30 ± 2 °C for 1 week. Then, the Trichoderma inocula were adjusted to 3.6 × 108 propagules/ml by using a haemocytometer slide. Then a mixture of MEM and Trichoderma propagules was applied (Abd-El-Khair et al. 2010).

For the preparation of rhizobateria, the pure culture of each bacterium was grown in nutrient broth medium at 30 ± 2 °C for 48 h. For mass production, 1-l conical flasks containing 500 ml of nutrient glucose (2%) broth medium (NGBM; 3 g beef extract, 5 g peptone, 20 g glucose, in 1000 ml distilled water and pH at 7.2) were autoclaved. Then, each flask was separately inoculated with 1.0 ml of broth culture of each bacterium. Then, the inoculated flasks were incubated at 30 ± 2 °C for 48 h and the flasks were shaken twice daily. Each bacterium inoculum was adjusted at 107–109 colony forming unit (CFU)/ml using dilution method. Each inoculum of bacterial species was applied as mixture of bacterial cells and cultural filtrate (El-Nagdi et al. 2019).

Greenhouse experiments

Three experiments were carried out in the greenhouse of Botany Department, National Research Centre, Egypt. The first experiment was conducted to study the ability of tested bio-control agents for improving growth parameters of faba bean, while the second and third ones were conducted to assess the pathogenic effect of tested bio-control agents against O. crenata. The soil free of O. crenata contamination was applied in the first experiment, while heavily contaminated soil artificially infected with a seeds bank of O. crenata (at the rate of 5% w/w) was applied in the 2nd and 3rd experiments.

The plastic pots (30-cm diameter), containing 5 kg of a sterilized mixture of loamy soil were arranged according to a completely randomized design on a bench under greenhouse conditions. In experiment I, the treatments were as follows: (1) Untreated control, (2) MEM only, (3) T. harzianum, (4) T. viride, (5) T. vierns, (6) NGBM only, (7) P. fluorescens, (8) B. subtilis and (9) B. pumilus. All treatments were applied at a rate of 50 ml microbial medium per pot before 1 week of sowing and then all pots were watered. In experiments II and III, pots filled with heavily contaminated soil artificially infected with a seeds bank of O. crenata (at a rate of 5% w/w) was applied and the microbial treatments were as follows: (1) O. crenata only, (2) O. crenata + glyphosate only, (3) O. crenata + NGBM only, (4) O. crenata + P. fluorescens, (5) O. crenata + B. subtilis, (6) O. crenata + B. pumilus, (7) O. crenata + MEM only, (8) O. crenata + T. harzianum, (9) O. crenata + T. viride and (10) O. crenata + T. vierns. All treatments were applied at a rate of 50 ml of microbial medium per pot in experiments I and II, while at 100 ml of microbial medium per pot in experiment III before 1 week of sowing and then all pots were watered. Eight seeds of faba bean (Giza 3 cv.) were sown at 3 cm from the soil surface of each pot and then the resulted plants thinned to 4 plants per pot (El-Dabaa et al. 2019). Eight pots were used as replicates for each treatment as well as the controls.

The growth parameters of faba bean plants as shoot length, shoot fresh and dry weights and leaf numbers were recorded in the first experiment at 2, 4 and 6 months of application. Effects of tested bio-control agents in reducing numbers, fresh and dry weights and length of the emerged juvenile O. crenata were recorded at 2 and 4 months and 2, 4 and 6 months of applications in experiments II and III, respectively. The same growth parameters of faba bean plants were also recorded at the same periods.

Statistical analysis

Data were subjected to analysis of variance using Computer Statistical Package User Manual Version 3.03, Barkley Co., USA, and mean values of each treatment was compared according to Duncan’s multiple range test at P = 0.05 level of significance (Snedecor and Cochran 1999).

Results

Experiment I

Effects of rhizobacteria and Trichoderma spp on the growth parameters of faba bean plants without O. crenata infection in pots

Effects of P. flourencens, B. subtilis, B. pumilus, T. harzianum, T. viride and T. vierns on the growth parameters of faba bean plants without O. crenata infection in pots are listed in Table 1. After 2 months, application of rhizobacteria treatments enhanced the shoot length (SL), fresh shoot weight (FSW), dry shoot weight (DSW) and leaf number (LN) in the ranges of 4–8%, 24–35%, 23–34% and 21–35% comparing with the controls, respectively. Maximum enhancement effects on FSW, DSW and LN were obtained by B. pumilus, whereas B. subtilis highly increased the SL. Tichoderma spp. increased the same growth parameters in the ranges of 6–9%, 16–31%, 10–32% and 16–32%, respectively. The highest increase in the previously mentioned parameters was obtained by T. vierns (SL, FSW and DSW) and T. viride (LN), respectively. After 4 months, the growth parameters mentioned before increased by rhizobacteria applications in the ranges of 22–27%, 104–131%, 99–124% and 29–52%, respectively. P. flourencens highly enhanced SL, FSW and DSW, while B. pumilus highly increased LN. Trichoderma spp. improved the growth parameters in the ranges of 14–31%, 43–112%, 32–105% and 27–34%, respectively. T. vierns highly increased the SL, FSW and DSW, while T. viride highly increased LN. After 6 months, rhizobacteria application increased the growth parameters in the ranges of 17–31%, 7–17%, 33–43% and 11–35%, while the increase with Trichoderma spp. was in the ranges of 22–28%, 12–53%, 41–104% and 20–64%, respectively. B. subtilis highly improved SL, FSW and DSW, while P. fluorescens increased LN. On the other hand, T. vierns, followed by T viride and T. harzianum showed the best growth parameters (Table 1).

Table 1 Effects of plant growth-promoting bacteria and Trichoderma species on growth parameters of faba bean without Orobanche crenata treatment under greenhouse conditions (Experiment I)
Full size table

Experiment II

Effect of rhizobacteria and Trichoderma spp. on Orobanche crenata parameters

The tested rhizobacteria species as well as Trichoderma spp. reduced the O. crenata juvenile parameters in pots, under artificial inoculation conditions, as shown in Table 2. After 2 months of application, the rhizobacteria reduced the juvenile numbers (JN) and juvenile fresh weight (JFW) and juvenile dry weight (JDW) in the ranges of 64–75%, 22–61% and 29–57%, compared with control corresponded with the values of 57, 41 and 57% with glyphosate treatment, respectively. B. pumilus highly reduced JFW and JDW, whereas P. fluorescens highly reduced JN. Many of the previously mentioned parameters were reduced by Tichoderma spp. in the ranges of 59–82%, 26–37% and 29–43%, compared to 57, 41 and 57 with glyphosate, respectively. T. vierns highly reduced the JFW and JDW, while T. harzanium highly reduced the JN, respectively. After 4 months, rhizobacteria application reduced many of the parameters mentioned before in the ranges of 43–60%, 40–55%, 41–52% and 38–52% compared to 68, 65, 67 and 49% with glyphosate, respectively. P. flourecenss highly reduced JN, JDW and juvenile length (JL), while B. subtilis highly reduced the JFW. Trichoderma spp. reduced the juvenile parameters in the ranges of 38–51%, 43–60%, 41–59% and 37–49%, compared to 68, 65, 67 and 49% with glyphosate, respectively. T. vierns highly exhibited fresh weight, dry weight and length of juvenile, respectively (Table 2).

Table 2 Effects of plant growth-promoting bacteria and Trichoderma species on O. crenata growth parameters in faba bean under greenhouse conditions (Experiment II)
Full size table

Effect on growth parameters of faba bean

Results in Table 3 revealed that P. flourencens, B. subtilis and B. pumilus increased the SL, FSW, DSW and LN of faba bean, under artificial inoculation conditions, in the ranges of 10–25%, 6–31%, 6–31% and 27–46% after 2 months of application, respectively. B. pumilus showed the best increase of SL, FSW and DSW, followed by B. subtilis and P. fluorsecens, while B. pumilus highly increased LN, followed by P. fluorsecens and B. subtilis. Whereas, T. harzianum, T.viride and T. vierns increased the same parameters in the ranges of 13–24%, 13–30%, 5–29% and 13–34%, compared to increase of 15, 64, 26 and 36% with glyphosate, respectively. Data showed that T. viride highly increased SL, FSW and DSW, while T. vierns highly increased LN. After 4 months of application, the rhizobacteria increased the SL, FSW, DSW and LN in the ranges of 13–24%, 9–15%, 11–15% and 7–38%, compared to 24, 37, 37 and 30% with glyphosate, respectively. Maximum increase in SL character was obtained by P. fluorescens, whereas maximum values of FSW, DSW and LN criteria were obtained by B. subtilis. Trichoderma spp. also improved the growth parameters in the ranges of 12–38%, 4–75%, 12–75% and 11–26%, compared to 24, 37, 37 and 30% with glyphosate, respectively. Results showed that T. vierns highly increased SL, FSW and DSW, while T. viride highly increased LN, respectively (Table 3).

Table 3 Effects of plant growth-promoting bacteria and Trichoderma species on growth parameters of faba bean with O. crenata infection, under greenhouse conditions (Experiment II)
Full size table

Experiment III

Effect of rhizobacteria and Trichoderma spp. on O. crenata parameters

Application of rhizobacteria species and Trichoderma spp. tended to produce significant reduction effect on O. crenata infestation as shown in Table 4. After 2 months of application, rhizobacteria reduced JN, JFW and JDW in the ranges of 31–54%, 9–50% and 30–60%, compared to values of 15, 4 and 20% with glyphosate, respectively. B. pumilus had the best reduction in JN and JFW, whereas B. subtilis highly reduced JDW, respectively. Tichoderma spp. reduced O. crenata juvenile parameters in the ranges of 31–92%, 7–86% and 50–80% compared to 15, 4 and 20% with glyphosate, respectively. T. viride highly reduced O. crenata juvenile parameters than other Trichoderma spp. After 4 months of application, rhizobacteria reduced the juvenile parameters in the ranges of 11–65%, 66–76%, 65–74% and 5–31%, compared to 92, 36, 37 and 43% with glyphosate, respectively. B. pumilus highly reduced JFW, JDW and JL, while B. subtilis highly reduced JN. The juvenile parameters’ reduction were in the ranges of 6–63%, 50–62%, 51–63% and 8–31% with Trichoderma spp., compared to 92, 36, 37 and 43% with glyphosate, respectively. T. vierns highly reduced the JFW and JDW, while T. harzianum highly reduced the JN and JL. After 6 months of application, the tested rhizobacteria reduced the juvenile parameters in the ranges of 23–64%, 42–65%, 25–64% and 12–65%, compared to 78, 72, 69 and 71% with glyphosate, respectively. P. fluorescens highly reduced O. crenata parameters than other rhizobacteria. Trichoderma spp. reduced the juvenile parameters in the ranges of 6–42-66%, 47–57%, 19–46% and 21–22%, compared to 78, 72, 69 and 71% with glyphosate, respectively. T. vierns highly reduced the JN and JFW, while T. harzianum highly reduced the JDW and JL, respectively (Table 4).

Table 4 Effects of plant growth-promoting bacteria and Trichoderma species on O. crenata growth parameters in faba bean under greenhouse conditions (Experiment III)
Full size table

Effect on growth parameters of faba bean

Under artificial inoculation conditions with O. crenata, rhizobacteria species and Trichoderma spp. increased the growth parameters of faba bean in pots as shown in Table 5. After 2 months of application, rhizobacteria increased the SL, FSW, DSW and LN in the ranges of 22–38%, 7–51%, 11–53% and 1–31%, while the increases were in the ranges of 19–33%, 22–64 and 23–32% and 7–11% with Tichoderma spp., compared to 16, 5, 7 and 2% with glyphosate, respectively. Results revealed that B. pumilus highly increased the growth parameters, followed by P. flourencens and B. subtilis. T. vierns highly increased the SL, FSW and DSW, while T. harzianum highly increased LN. After 4 months of application, the rizhobacteria increased the growth parameters in the ranges of 5–17%, 12–46%, 15–53% and 9–23%, while Trichoderma spp. improved it in the ranges of 6–19%, 11–38%, 16–44% and 5–31%, compared to 2, 9, 5 and 6% with glyphosate, respectively. B. pumilus had the highest increase of growth parameters. T. vierns highly increased growth parameters, followed by T. harzianium and T. viride, respectively. After 6 months of inoculation, the rhizobacteria increased the growth parameters in the ranges of 13–21%, 36–43%, 13–24% and 14–30%, while the growth parameters were improved in the ranges of 12–25%, 18–42%, 3–23% and 8–10% with Trichoderma spp., compared to 5, 23, 6 and 6% with glyphosate, respectively. B. pumilus highly improved the SL, FSW and DSW, while P. fluorescens increased leaf number. T. viride highly increased growth parameters, than other Trichoderma spp., respectively (Table 5).

Table 5 Effects of plant growth-promoting bacteria and Trichoderma species on growth parameters of faba bean with O. crenata infection, under greenhouse conditions (Experiment III)
Full size table

Discussion

Broomrape is a parasitic weed which decreases the production of legumes, especially faba bean (Vicia faba; Rubiales et al. 2016). It is a root holoparasitic plant devoid of chlorophyll and entirely depending on the host for nutritional requirements (Habimana et al. 2014). Application of biotic agents can induce systemic resistance in treated plants against many plant diseases (Walters et al. 2013). Therefore, application of chemical control with systemic herbicides (e.g. glyphosate) at low rates as well as application of fumigation, solarization and cultural practices is possible for controlling broomrape infection, where use of economical and environmentally friendly control is very important (Rubiales and Aparicio 2012). In this study, P. flourencens, B. subtilis, B. pumilus, T. harzianum, T. viride and T. vierns were applied in three separate pot experiments for determining their ability to enhance growth parameters of faba bean as well as decreasing O. crenata juvenile parameters. Table 2 illustrates the effect of rhizobacteria on O. crenata. Results of experiment II revealed that the tested rhizobacteria and Trichoderma could protect faba bean plants for 4 months against O. crenata infection, where most plants were dead after that. The rhizobacteria and Trichoderma spp. had better reduction of JN than glyphosate after 2 months of application, while both B. subtilis and B. pumilus had the highest reduction of JFW; their effect was equal to the herbicide for JDW. Effects of bio-control agents were high until the 4th month, but it was less than the herbicide effect. In experiment III, the tested bio-control agents highly reduced the juvenile parameters after 2 months, while it reduced the JFW and JDW after 4 months more than the herbicide, respectively. The tested bio-control agents were effective until 6 months, but their effect was less than that of the herbicide. These results are in agreement with those recorded by Samejima and Sugimoto (2018) who reported that the weeds can be controlled by using resistant or tolerant varieties, microbiological approach, cultural practices, chemical controls and integrated management. Boari and Vurro (2004) revealed that numerous microorganisms potentially useful for bio-controlling of Orobanche species were isolated and reported, but none had been subjected to continuous widespread use. Improving soil fertility also by using beneficial microorganisms appeared to decrease O. crenata infestation and its suppressiveness effects on host growth (Elabaied et al. 2018). Application of T. harzianum, T. viride and T. hamatum reduced the number of Orobanche shoots in comparison with the control (Nawar and Sahab 2011; Hyder et al. 2017).

The results cleared that P. flourencens, B. subtilis, B. pumilus, T. harzianum, T.viride and T. vierns could improve the growth parameters of faba bean plants, i.e. shoot length, shoot fresh weight, shoot dry weight and leaf number, when applied with or without Orbanche spp. The plant growth-promoting rhizobacteria (PGPR) are rhizospheric microbes produce bioactive substances and promote plant growth and/or protect them against pathogens. Root-colonizing bacteria also improved the plant growth through direct stimulation by producing growth regulators or by suppression of pathogens. PGPR have to be highly competitive to successfully colonize the root zone (Raaijimaker et al. 2002). These results are in agreement with those recorded by Abd-El-Khair et al. (2019) who mentioned that B. pumilus, followed by B. subtilis and P. fluorescens had the highest increase in plant growth parameters in cowpea, respectively. Application of B. subtilis and B. pumilus, alone or in combination, controlled root-knot nematode and significantly increased the growth parameters of pea plants such as shoot length, shoot fresh and dry weights, leaf numbers and pod fresh and dry weights in pot experiment (El-Nagdi et al. 2018).

The antagonistic potential effects of both T. harzianum (Plantgard) and B. subtilis (Rhizo-N) controlled the incidence of Fusarium root rot disease and increased the growth parameters per plant viz., shoot length, both fresh and dry shoot weights, branch number, root length and both fresh and dry root weights as well as yield parameters as pod number and fresh and dry weights of pod in faba bean plants in field application in organic farming (Abd-El-Khair et al. 2018). The herbicide Clethodim and three Trichoderma strains, as lone or in combination application controlled weeds, root-knot nematodes and Rhizoctonia root rot in field applications. The plant growth parameters, (i.e. shoot length, shoot fresh and dry weights and branch and leaf numbers) and yield parameters (fresh pod and dry weights, seed number per pod, seed weight and ash pod weight of plant) were greatly improved with combination treatments when compared to each treatment separately (El-Dabba et al. 2019).

Conclusion

  • Broomrape is a weed causing great damage to legumes specially faba bean.

  • Several methods were applied to control broomrape.

  • This study aimed to apply Trichoderma spp. as well as three rhizobacteria species in comparison to herbicidal effect of glyphosate for controlling broomrape infesting faba bean.

  • Results revealed that the application of Trichoderma spp. as well as rhizobacteria species could play an important role in controlling broomrape in faba bean as a natural bioherbicide as well as enhancing the growth parameters of faba bean.