INTRODUCTION

Plants form symbiotic associations with numerous plant growth-promoting rhizobacteria (PGPR) species, deriving a multitude of growth and protection benefits. Many of these species improve plant biomass, augment flowering and enable toleration of biotic and abiotic stresses (Lugtenberg & Kamilova, 2009). Species of Bacillus in particular are considered to have an important role in the development of sustainable agricultural systems by providing benefits to plant growth (Dhayalan & Karuppasamy, 2021). For instance, B. cereus Frankland & Frankland is an effective root colonizer which can suppress plant diseases (Shameer & Prasad, 2018) and enhance biomass in different plants (Dutta et al., 2013), as can B. subtilis (Ehrenberg) (Blake et al., 2021) and B. amyloliquefaciens Priest (Ngalimat et al., 2021). Synthesis of various bioactive molecules that have a wide spectrum of anti-pathogenic activities and effective rhizosphere competence for nutrients and niches are some of the many attributes that contribute to the success of B. cereus, B. subtilis and B. amyloliquefaciens as effective suppressors of various plant diseases (Ali et al., 2020). However, relatively little is known about how these three ubiquitous soil bacterial species extend their effects up to foliage-feeding insects and their natural enemies (Gange et al., 2012).

Aphids, including cabbage aphid (Brevicoryne brassicae L.), green peach aphid (Myzus persicae [Sulzer]) and mustard aphid (Lipaphis erysimi [Kaltenbach]) reduce the biomass and quality of many Brassicaceae plants on a large scale and spread epidemic viral diseases, throughout the world (Blackman & Eastop, 2000). B. brassicae is a specialist aphid that causes curling, distortion and yellowing of foliage. In severe infestations, damage can be seen in the inner core of cabbage heads and plants may completely wither and die. Furthermore, B. brassicae is a vector of many viral diseases such as cauliflower and turnip mosaic, cabbage black ringspot and cabbage ring necrosis (Broadbent, 2015). M. persicae is a generalist feeder that attacks a wide range of plants worldwide and transmits over 100 viral diseases (Gadhave et al., 2020). L. erysimi is also one of the most damaging pests of brassicas and is a vector of several viral diseases. Depending upon plant species, plant growth stage and the extent of infestation, L. erysimi can cause up to 90% loss in some crop plants (Patel et al., 2004).

There is some evidence that B. cereus, B. subtilis and B. amyloliquefaciens can reduce the growth, reproduction and population build up of B. brassicae on calabrese (Brassica oleracea L.) in both laboratory and field conditions (Gadhave, Finch, et al., 2016a; Gadhave & Gange, 2016), though effects varied between bacterial species. Meanwhile, a mixture of other PGPR species (including Pseudomonas, Azotobacter and Azospirillum) reduced fecundity of B. brassicae feeding on canola Brassica napus L. in glasshouse conditions (Nasab et al., 2019). In contrast, the effects of PGPR on M. persicae appear to be more variable. Application of B. cereus reduced aphid fecundity on bell pepper (Capsicum annuum L.) in a glasshouse (Mardani-Talaee et al., 2017), but this species and B. amyloliquefaciens had no effect on aphids on the same plant in field conditions (Herman et al., 2008). Meanwhile, another PGPR, Pseudomonas fluorescens (Flügge) increased the growth of M. persicae but had no effect on B. brassicae (Pineda et al., 2012). To date, there have been no studies of the effects of PGPR applications on L. erysimi.

Taken together, these variable results show that PGPR-aphid interactions are likely to be context dependent, with outcomes determined by the identity of the PGPR, the target insect, the conditions in which the experiments take place and the composition of the indigenous bacterial community (Gadhave, Hourston, et al., 2016b; Gadhave et al., 2018). Given that the interactions between PGPR and other foliar-feeding insects are equally variable (Friman et al., 2021), there is a clear need for more studies that might enable us to search for patterns and thereby unravel these complex interactions. Doing so is critical if PGPR are to fully realize their potential as alternative forms of pest control in sustainable agriculture (Basu et al., 2021).

Studies of the effects of PGPR on predators and parasitoids of herbivorous insects are rarer but show as much variation as those with the herbivores themselves. A laboratory study showed that the volatile blend of Arabidopsis thaliana (L.) Heynh. plants treated with P. fluorescens was less attractive to Diaeretiella rapae (McIntosh) (Hymenoptera: Braconidae), thereby reducing parasitism of M. persicae (Pineda et al., 2013). However, parasitism of Mamestra brassicae L. larvae by another Braconid, Microplitis mediator (Haliday), was increased when feeding on the same plant when treated with this bacterium (Pangesti et al., 2015). Meanwhile, B. cereus and B. subtilis were found to increase rates of parasitism of B. brassicae by D. rapae (McIntosh), through density-dependent responses (Gadhave, Finch, et al., 2016a).

Studies of PGPR effects on predators of herbivorous insects are even rarer but equally inconclusive. For example, Boutard-Hunt et al. (2009) found limited evidence that natural enemy numbers (not specified, but including Coccinellidae, Neuroptera, Diptera and Anthocoridae) were higher on pepper plants treated with B. amyloliquefaciens. Saravanakumar et al. (2008) found that field applications of Pseudomonas to rice reduced leaffolder, Cnaphalocrocis medinalis (Guenée) incidence and lured its natural enemies, whereas Gadhave, Finch, et al. (2016a) found no effect of the aforementioned Bacillus species on predators (Coccinellids and Syrphid larvae) of B. brassicae in field-grown calabrese.

The aim of this field experiment, conducted in the subtropical climate of India, was to investigate the effects of seed application of B. cereus, B. subtilis and B. amyloliquefaciens on the growth of calabrese and its infestation by the aphids B. brassicae, M. persicae, L. erysimi and colonization by their most abundant natural enemies; D. rapae and syrphid fly larvae. We also recorded attack by flea beetles (Phyllotreta cruciferae [Goeze]), diamondback moth larvae (Plutella xylostella L.) and cabbage whitefly (Aleyrodes proletella L.) over the course of one growing season. The plant biomass data were given in Gange and Gadhave (2018), and the insect data are reported here. Application of B. amyloliquefaciens and a mixture of B. amyloliquefaciens, B. cereus and B. subtilis increased the yield of plants by 48% and 70%, respectively, whereas all bacterial treatments significantly increased the variability in the size of plants, relative to controls (Gange & Gadhave, 2018).

It is a fundamental principle of science that experiments should be repeatable (Baker, 2016), something which is particularly important in the soil environment (Bond-Lamberty et al., 2016). The work reported here was designed to be similar to our previous study (Gadhave, Finch, et al., 2016a) on a Bacillus-calabrese-cabbage aphid-natural enemy model system which was conducted in temperate conditions (UK). As the effects of PGPR on plants and herbivores are often strain specific (Dhayalan & Karuppasamy, 2021), it is important to know if the effects seen in that study (above) are context specific or whether other locally isolated strains of similar Bacillus spp. also have detrimental effects on insects in a different climate such as the tropics, where local soil microbial communities would differ. The current study differs from that of Gadhave, Finch, et al. (2016a) because treatments were applied only to the seeds, to determine if this simple application method could influence levels of pests and their natural enemies in field conditions. Due to the limited shelf life of liquid products in warm climates, seed coating is currently the most viable method of PGPR delivery to roots (Arriel-Elias et al., 2018). This has relevance in determining the efficacy and applicability of using bacterial seed coating technology, which is becoming increasingly popular for its field applications (Rocha et al., 2019). We have already shown that some seed applications, particularly a mix of B. cereus, B. subtilis and B. amyloliquefaciens, resulted in increased root and shoot biomass in the tropical environment (Gange & Gadhave, 2018). Here, we hypothesized that the colonization of roots with plant growth-promoting Bacillus spp. would facilitate an array of additional benefits to plants via suppressed aphid numbers and Bacillus-mediated natural enemy responses.