Beneficial Insects Deliver Plant Growth-Promoting Bacterial Endophytes between Tomato Plants

Abstract

Beneficial insects and mites, including generalist predators of the family Miridae, are widely used in biocontrol programs against many crop pests, such as whiteflies, aphids, lepidopterans and mites. Mirid predators frequently complement their carnivore diet by feeding plant sap with their piercing-sucking mouthparts. This implies that mirids may act as vectors of phytopathogenic and beneficial microorganisms, such as plant growth-promoting bacterial endophytes. This work aimed at understanding the role of two beneficial mirids (Macrolophus pygmaeus and Nesidiocoris tenuis) in the acquisition and transmission of two plant growth-promoting bacteria, Paraburkholderia phytofirmans strain PsJN (PsJN) and Enterobacter sp. strain 32A (32A). Both bacterial strains were detected on the epicuticle and internal body of both mirids at the end of the mirid-mediated transmission. Moreover, both mirids were able to transmit PsJN and 32A between tomato plants and these bacterial strains could be re-isolated from tomato shoots after mirid-mediated transmission. In particular, PsJN and 32A endophytically colonised tomato plants and moved from the shoots to roots after mirid-mediated transmission. In conclusion, this study provided novel evidence for the acquisition and transmission of plant growth-promoting bacterial endophytes by beneficial mirids.

Keywords: Macrolophus pygmaeus; Nesidiocoris tenuis; beneficial mirids; plant growth-promoting bacterial endophytes.

Figure 1

Description of the experiment. Day 1, surface-disinfected seeds were transferred to Petri dishes containing 1% water agar and were incubated in a growth chamber to allow seed germination. Day 3, seeds were treated with sterile MgSO₄ (mock-inoculated) or inoculated with a bacterial suspension of Paraburkholderia phytofirmans PsJN (PsJN) or Enterobacter sp. 32A (32A) by overnight incubation in the growth chamber. Day 4, germinated seeds with the same root length were selected and each seed was transferred to a sterile glass tube containing half-strength Hoagland. Day 7, a freshly emerged mirid adult (Macrolophus pygmaeus or Nesidiocoris tenuis) was placed in each glass tube containing a tomato plant that was either mock-inoculated or inoculated with PsJN or 32A. Before transferring the mirid in the glass tube, shoot length was measured by image analysis. Tubes were incubated in the growth chamber in order to allow mirids to feed on tomato plants (acquisition period). Day 11, for double labelling of the oligonucleotide probes for fluorescence in situ hybridisation (DOPE-FISH) analysis, mirids were collected at the end of the acquisition period on mock-inoculated plants or plants inoculated with PsJN or 32A, shoot length was measured by image analysis, and whole plants were collected for bacterial re-isolation and fresh weight assessment. Each mirid was transferred to a new glass tube containing a mock-inoculated tomato plant and incubated to allow the mirids to feed on the tomato plants (mirid-mediated transmission), and then shoot length was measured. Day 14, mirids and plants after mirid-mediated transmission were collected for bacterial re-isolation, and the tomato shoot length and fresh weight were assessed.

Figure 2

Location of endophytic bacterial strains on mirids. Macrolophus pygmaeus (A,C,E) and Nesidiocoris tenuis (B,D,F) abdomen (a), thorax (b) and leg (c) samples were collected at the end of the acquisition period (Day 11) on plants inoculated with Paraburkholderia phytofirmans PsJN (PsJN) (A,B) or Enterobacter sp. 32A (32A) (C,D) or mock-inoculated plants (E,F). PsJN cells were hybridised with the EUBmix and Bphyt probes (A,B) and 32A cells were hybridised with the EUBmix and Gam42a probes (C,D). Mirids fed on mock-inoculated plants (E,F) were hybridised with the EUBmix and Gam42a probes. Five replicates (mirids) were analysed for each treatment and representative pictures were selected. Bars correspond to 10 µm.

Figure 3

Quantification of endophytic bacterial strains in tomato plants. Bacterial re-isolation was carried out from seed-inoculated whole plants at the end of the acquisition period (Day 11) and from shoots or roots of plants at the end of the mirid-mediated transmission (Day 14) with Macrolophus pygmaeus (A) or Nesidiocoris tenuis (B). The quantity of re-isolated bacteria are expressed as colony forming units (CFU) per gram of fresh weight of the whole plant (CFU g⁻¹) and plant shoot (CFU g⁻¹), or as CFU for each plant root (CFU root⁻¹) of mock-inoculated plants (Mock, green) and plants inoculated with Paraburkholderia phytofirmans PsJN (PsJN, red) or Enterobacter sp. 32A (32A, blue). The two-way analysis of variance showed no significant differences between the two experimental repetitions (p > 0.05) and data from the two experiments were pooled. Mean and standard error values for positive samples and at least nine replicates (plants) are presented for each treatment. For each treatment, no significant differences were found in the pairwise comparisons between PsJN- and 32A-inoculated samples, according to the Mann–Whitney test (p ≤ 0.05). Neither PsJN nor 32A bacterial colonies were isolated from the mock-inoculated samples.

Figure 4

Quantification of endophytic bacterial strains on the mirid epicuticle and internal body. Bacterial re-isolation was carried out from a mirid washing suspension to collect bacteria adhering to the mirid epicuticle or from tissue grinding of surface-disinfected mirids to collect bacteria in the mirid internal body at the end of the mirid-mediated transmission (Day 14) with Macrolophus pygmaeus (A) or Nesidiocoris tenuis (B). The quantity of re-isolated bacteria is expressed as the colony forming units per mirid (CFU mirid⁻¹) fed on mock-inoculated plants (Mock, green) and plants inoculated with Paraburkholderia phytofirmans PsJN (PsJN, red) or Enterobacter sp. 32A (32A, blue). The two-way analysis of variance showed no significant differences between the two experimental repetitions (p > 0.05) and data from the two experiments were pooled. Mean and standard error values for positive samples and at least nine replicates (mirids) are presented for each treatment. Asterisks indicate significant differences in the pairwise comparisons between the PsJN- and 32A-inoculated samples, according to the Mann–Whitney test (p ≤ 0.05). Neither PsJN nor 32A bacterial colonies were isolated from mirids fed on the mock-inoculated plants.

Figure 5

Effects of mirid feeding on tomato shoot length. Changes in shoot length caused by Macrolophus pygmaeus (A,B) or Nesidiocoris tenuis (C,D) feeding were assessed on mock-inoculated plants (Mock) and plants inoculated with Paraburkholderia phytofirmans PsJN (PsJN) or Enterobacter sp. 32A (32A) and calculated as the difference between the shoot length measured before (Day 7) and after (Day 11) the acquisition period (A,C), or before (Day 11) and after (Day 14) the mirid-mediated transmission (B,D). The two-way analysis of variance showed no significant differences between the two experimental repetitions (p > 0.05) and data from the two experiments were pooled. Mean and standard error values for positive samples and at least nine replicates (plants) are presented for each treatment. Different letters indicate significant differences among treatments according to Tukey’s test (p ≤ 0.05). Asterisks indicate significant differences in the pairwise comparisons between the mock-inoculated and bacterium-inoculated plants, according to Student’s t-test (p ≤ 0.05).