Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) and Southern Corn Rootworm (Diabrotica undecimpunctata howardi Barber) Identified as Vectors of Late-Season Decline Disease-Causing Pantoea ananatis

Abstract

Pantoea ananatis was recently described as the causative agent of late-season decline, a new bacterial disease first observed affecting field corn plants, in the Texas Panhandle. The rapid spread of the disease throughout the region and the patchy distribution of symptomatic plants in affected fields, as well as routine observations of edge effects, in which plants with severe symptoms are observed on the edges of affected fields, led us to hypothesize that vectors might be involved in the dissemination of the disease pathogen. In this study, we investigated the western corn rootworm (Diabrotica virgifera virgifera LeConte) and southern corn rootworm (Diabrotica undecimpunctata howardi Barber) for any naturally occurring association with P. ananatis and potential to acquire and transmit the bacterial pathogen. Additionally, we investigated the transgenic corn encoding insecticidal Bacillus thuringiensis proteins (Bt) pyramided with RNAi interference anti-rootworm technology for its potential to protect against any larval role in the transmission of the pathogen through their feeding activities on corn roots. We successfully recovered naturally occurring P. ananatis from samples of both rootworm species collected from corn plants in the field. Following acquisition assays, the acquired pathogen was successfully recovered from previously P. ananatis-free adult rootworms, their eggs, as well as first-instar larvae, suggesting an affinity of the bacteria to establish an endosymbiotic and transovarial association with both rootworm species. Additionally, the transgenic Bt corn with RNAi anti-rootworm technology was ineffective in preventing the transmission of the pathogen by the infected larvae. Findings from this study confirm a vector role in the transmission of the disease pathogen.

1. Introduction

In the United States, corn (Zea mays L.) is the most important agricultural crop with a 2023 grain value of $73.8 billion from a total of 94.6 million acres [1]. Consequently, any factor, biotic or abiotic, with the potential to cause reductions in yields from cereal production, poses a threat to the availability of the basic sources of both food and feed dietary calories. In 2023, late-season decline (LSD), a new bacterial disease affecting corn caused by Pantoea ananatis [2] was reported in Texas by Obasa et al. [3]. In severe outbreaks, yield loss from the disease can exceed ninety percent [3]. In 2024, LSD was identified, for the first time, affecting sorghum in Texas [4]. In both cases, the disease quickly became widespread following the original discoveries. The disease is often associated with a random patchy distribution of symptomatic plants in affected fields (Figure 1a), as well as the presence of edge effects in which plants with severe symptoms are observed on the edges of affected fields (Figure 1b). Both attributes are also characteristic of insect-vectored diseases. Therefore, to better understand the LSD disease pathogenesis, we sought to investigate the possible role of insect vector(s) in the disease epidemiology. The need to better understand the factors that mediate the spread of P. ananatis-associated disease is further underscored by recent publications of new reports of diseases caused by P. ananatis in wheat [5,6,7], corn [8], rice [9,10,11], and onion [12,13].

To explore our vector hypothesis, two candidate vectors—western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) and southern corn rootworm (SCR; Diabrotica undecimpunctata howardi Barber), were selected for investigation in the current study based on the following criteria: SCR larvae have been reported to be a minor vector of Stewart’s wilt disease in corn caused by Pantoea stewartii [14], bacterial wilt caused by Erwinia tracheiphila in cucurbits [15], in addition to squash mosaic virus, and muskmelon necrotic spot virus also in cucurbits [15]. SCR actively feeds on sorghum and wheat, which are also hosts of P. ananatis [4,6], and is routinely observed in corn fields, including those with LSD infections, and consequently, may facilitate the spread of the disease pathogen within and across fields with LSD susceptible host crops. Moreover, in one case of a severe LSD infection in a commercial corn field in 2023 in Armstrong County, Texas, heavy infestation of adult WCR was observed at corn reproductive stages (R1-R2: silk). At that point, the disease symptomatology was largely at advanced stages, showing necrosis of the three to four uppermost leaves and sterile tassels. But the fact that the disease undergoes a characteristic progression of symptoms that takes time to become fully expressed [3], and that adult WCR emerges just right before tasseling and at reproductive corn stages, suggests that if WCR vectored LSD, the adult stage likely was not involved in the initial transmission that resulted in the advanced stage symptoms observed in the field. In contrast, rootworm larvae feeding on corn roots and transmitting the pathogen, with the disease progressing at early corn stages and fully expressing the symptoms at reproductive stages, is a more plausible explanation for the observed scenario. However, this does not rule out that adult WCR that have already acquired the pathogen might also be transmitting it within the same field. This scenario is plausible, additionally, given that adult WCR has been reported to vector P. ananatis in the case of the white spot disease of corn [8]. Rootworms lay eggs in the soil, and their larvae damage plant roots at vegetative stages in the current or following season, depending on the species. Lesions inflicted by rootworm larvae on host roots affect the ability of infested plants to absorb water and nutrients from the soil and can serve as an entry point for pathogens at early stages of crop development [16].

Corn, sorghum, and wheat are major crops produced in the Texas Panhandle, a region of Texas with confirmed cases of LSD, and this suggests that the likelihood of SCR and WCR being exposed to the LSD pathogen is high. Although adult corn rootworms have already been implicated in the transmission of P. ananatis in corn [8], there are limited reports involving the larval stage of the pest vectoring plant pathogens. A study was conducted to determine the possible role of corn rootworms in the transmission of LSD in corn. The specific objectives of this study were to (1) sample and test natural populations of both WCR and SCR from LSD-infected fields for the presence of endosymbiotic P. ananatis that share identity with those isolated from symptomatic plants within the same field, (2) investigate vertical transmission of P. ananatis from adult rootworms to their respective offspring, and (3) investigate transmissibility of P. ananatis to healthy corn plants infested with P. ananatis-infected rootworm larvae.

2. Materials and Methods

Bacterial isolation, purification, and identification: Bacterial isolations from LSD symptomatic corn leaf tissues arising from natural infections were done as previously described [3]. Briefly, symptomatic tissues were rinsed in sterile-distilled water, surface sterilized in ten percent hypochlorite solution for one minute, then rinsed in five changes of sterile distilled water and blotted dry. The surface-sterilized tissues were then cut into 0.5–1 cm-long sections with sterile scalpel blades, and the cut sections were placed in Petri dishes containing Luria Bertani (LB) agar. For isolations from rootworms, three adult rootworms, fifty rootworm eggs, or fifteen first-instar rootworm larvae were similarly surface-sterilized in 10 percent hypochlorite solution, then disrupted in a 2-mL tube containing 0.5-mm-diameter zirconia/silica beads (Biospec, Bartlesville, OK, USA) and 500 µL of LB broth using an MP-Bio FastPrep-24 5G (MP Biosciences, Irvine, CA, USA) bead-beater in three cycles at speed 4.0 for 45 s. An aliquot of a 10× dilution of the supernatant was subsequently plated on LB agar in a Petri dish, and the plates were incubated in the dark at 30 °C to obtain bacterial colonies. Following the incubation period, bacterial outgrowth from the sectioned tissue samples, or resultant colonies from rootworm preparations, were purified through three successive rounds of single colony streaking onto LB agar. Representative single, round, and yellowish bacterial colonies with smooth margins from the final purification rounds were transferred separately into LB broth and incubated overnight at 30 °C and 240 rpm. Genomic DNA was extracted, as previously described by Maniatis et al. [17], from overnight cultures and used in downstream analysis. Amplification of the 16S rRNA by polymerase chain reaction (PCR) was carried out using the primer pair 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) [18] at a final concentration of 0.5 µM and 1× final concentration of Bio-rad 2× HF Master-Mix (BIO-RAD, Hercules, CA, USA) in a 25 µL total reaction volume. The PCR amplification cycle consisted of an initial denaturing step at 98 °C for 3 min, 35 cycles of three steps consisting of 98 °C for 10 s, 57 °C for 30 s, 72 °C for 30 s, and a final step of 72 °C for 10 min. The PCR products, ~1500 bp amplicons, were sequenced, and the resulting sequences were used to identify the respective bacteria through BLAST (version 2.17.0) searches.

Western and Southern Corn Rootworm Rearing Procedures. Non-diapausing WCR and SCR adults were sourced from Crop Characteristics, Inc. (Farmington, MN, USA). Once in the laboratory, insects were transferred into cages (30.48 cm × 30.48 cm × 30.48 cm) (Restcloud, purchased through Amazon, USA—https://www.amazon.com/) separated by species. The insect colonies were fed slices of tender sweet corn ears and an adult dry artificial diet supplement formulated for each species (Frontier Agricultural Sciences, Newark, DE, USA) in 35 mm × 10 mm Petri dishes (Global Industrial, Port Washington, NY, USA). The corn ear slices and dry diet were replaced twice and once every week, respectively. Water was also provided for insects in 96 mL plastic soufflé cups with lids covering the water to prevent evaporation (Dart Container Corporation, Mason, MI, USA). The plastic lids were perforated in the center using a paper single-hole puncher, and a medium braided cotton roll (Richmond Dental & Medical, Charlotte, NC, USA) was inserted through the lid and immersed in the water. The cotton roll absorbs water by superficial tension and serves as a wick for adult rootworms to drink water. The water and dispensers were replaced weekly. Rootworm colonies were maintained in an environmentally controlled incubator (Percival Scientific, Perry, IA, USA) at 26 °C, ~60% relative humidity, 14 h:10 h (light: day) photoperiod, and 290 lumens throughout the experiment. Dead insects, resulting from natural causes and/or stress during shipment from the commercial provider, were removed from cages as needed while the rootworms acclimated to the new laboratory rearing environmental conditions, and to reduce the possibility of entomopathogens spreading within the colonies.

Rootworm Bacterial Acquisition Assay. Before experimental use, five randomly selected adult rootworm samples were tested for the presence of endosymbiotic Pantoea sp. as described above. Subsequently, the respective rootworm colonies were subdivided into two cages per species, with approximately four hundred individuals (~1♂:1♀ ratio) per cage. The cages were labeled as 1 and 2 for WCR, 3 and 4 for SCR. Corn ear slices and water dispensers from cages 1 and 3 were removed from the cages to preclude these colonies from water intake. After two days, sweet corn ear slices, impregnated by soaking for 2 h in the suspension (OD₆₀₀ = 0.2) of the late-season decline-inducing bacterium, P. ananatis isolate B623 [3] maintained in the laboratory at −80 °C, were placed in weighing boats and introduced into cages 1 and 3 to substitute for water in the dietary intakes in the respective cages. The bacteria-infused corn ear slices were left in the cages until dried (~6–7 days) and replaced with freshly prepared bacteria-infused ear slices two additional times during a four-week period. Finally, the provision of fresh non-treated corn ear slices was resumed in cages 1 and 3 as before and continued for the duration of the experiment. Successful acquisition of the bacteria resulted in the generation of WCR^B623+ and SCR^B623+. Insects in cages 2 and 4 continued to receive non-bacteria-infused corn ear slices and the dry dietary supplement and served as controls, WCR^B623− and SCR^B623−, respectively. A week after the withdrawal of the bacteria-infused corn ear slices from cages 1 and 3, three adult rootworms were randomly selected from each cage and, separately, tested for the presence of P. ananatis within their bodies as described above. When P. ananatis was recovered, the 16S rRNA sequence was aligned to that of P. ananatis isolate B623 for comparison.

Egg Collection and Larval Recovery. Egg and larval recovery were conducted following a slightly modified procedure of Chu et al. [19]. Briefly, 25 mL of 1% Drosophila agar solution (Fisher Scientific, Waltham, MA, USA) was made and dispensed in 100 × 15 mm Petri dishes (Global Industrial, Port Washington, NY, USA). After solidifying, filter paper (Fisher Scientific, Waltham, MA, USA) was placed on the agar. Four pieces of cheesecloth (Dritz, Nashville, TN, USA) were cut and placed over the filter paper inside the Petri dish. The dishes were subsequently introduced to all four adult rootworm cages as oviposition substrates. The agar helped to maintain humidity and prevent egg desiccation. Inside the cages, the oviposition plates were covered with a sheet of aluminum foil folded and molded to make a small “dome-like” structure over the plates to create a darkened environment for female rootworms to lay eggs on the oviposition substrate. Eggs were collected from the oviposition plates, separately, by cage number. To recover the eggs laid on the oviposition substrates, cheesecloths with eggs were immersed in a beaker containing 500 mL of water and gently swirled to dislodge the eggs. An 80-mesh sieve was next used to recover the dislodged eggs from the water in the beaker. The recovered eggs were rinsed with water several times inside the sieve and then transferred onto filter paper, which was in turn placed in a Petri dish containing 1% percent agar. To prevent mold growth, 6 to 8 droplets of 0.0475% methyl 1-(butyl carbamoyl)-2-benzimidazolecarbamate solution (Sigma-Aldrich, Inc., Burlington, MA, USA) were added onto the eggs using a disposable pipette. Thereafter, a small paintbrush was used to aid in the spreading of the eggs over the filter paper. A second filter paper was added to the underside of the Petri dish lid over the eggs to prevent the formation of condensate while maintaining humid conditions within the respective dishes. The egg incubation plates were labeled, placed in an incubator, and maintained at 26 °C, ~60% relative humidity, 14 h:10 h (light: day) photoperiod, and 290 lumens until the eggs hatched. First-instar larvae of WCR and SCR were recovered and used for the pathogen transmission bioassay.

Corn-Root Infestation and Pathogen Transmission Assay. Two field corn hybrids from existing stocks that were part of a separate and independent study were selected and grown for the experiment. The corn hybrids and transgenic insecticidal technology package produced per hybrid included (1) DKC107-33 having Cry3Bb1 + Gpp34/Tpp35Ab1 Bacillus thuringiensis proteins (Bt), pyramided with double-stranded RNA, DvSnf7 [20] designed to target the WCR Snf7 gene (RNAi interference technology) (hereafter Bt/RNAi+), and (2) DKC 67-72 encoding no belowground-Bt, nor dvSnf7 RNA (hereafter Bt/RNAi-). The corn hybrid DKC107-33 was included, in the event it was determined that WCR^B623+ and SCR^B623+ could transmit P. ananatis B623, to be able to evaluate the potential of the transgenic insecticidal technologies to mitigate against successful transmission of the acquired bacterium to healthy plants. The seeds of the different corn hybrids, which came coated with the insecticidal active ingredient clothianidin, were washed to remove the insecticide seed treatment and allow rootworm feeding. Briefly, corn seeds were rinsed in a ten percent hypochlorite solution for one minute with vortexing, which resulted in the removal of most of the seed treatment coating. The bleached and surface-disinfested seeds were then rinsed in five changes of sterile distilled water and subsequently blotted dry on Kimwipes (catalog #34155; Kimberly-Clark, Roswell, GA, USA). Thereafter, the seeds of the two corn hybrids were planted in Berger BM1 Nutrient Retention General Purpose Media (Hummert International, Topeka, KS, USA) in 1.5-gallon plastic pots at one plant per pot. The pots were lightly watered every other day and maintained at a 14-h photoperiod and average relative humidity and temperature of 80% and 30 °C, respectively, in a Vivosun GIY-SGS-44 Lite Smart Grow Tent (Vivosun, Ontario, Canada). At the V4–V5 growth stage, the roots of corn plants in three replicate pots were exposed, and ten neonate larvae of either bacteria-infected WCR or SCR were placed on exposed root hairs using a small paintbrush. Roots infested with the control larvae WCR^B623− or SCR^B623− served as checks. Because the seeds were previously washed, and the residual activity of clothianidin in corn lasts ~21 days after planting, under optimal growing conditions [21], any possible remaining insecticide after the seed wash step would have been fully spent by plants at the V4–V5 stage (~21–32 days after planting) and therefore have no impact on the larvae following root infestation. After infestation with the rootworm larvae, infested roots were lightly and gently covered up again with the potting media, and the topsoil was lightly misted with water using a portable handheld electric sprayer. Subsequently, the infested and control pots were grown separately in grow tents and maintained under the growth conditions described above, and monitored by visual observations for the characteristic stunting and foliar symptoms of LSD disease described by Obasa et al. [3] around the reproductive growth stage of the experimental plants. When symptoms were observed, symptomatic tissues were sampled and tested for the presence of P. ananatis isolate B623.

Qualitative data obtained from the transmission bioassay were analyzed using contingency tables to determine a possible association between plants exposed to rootworm larvae derived from the different acquisition treatments and the development of LSD symptoms. The binomial categories established for plants infested with rootworm larvae were (a) plants infested with either WCR^B623+ or SCR^B623+ or (b) WCR^B623− or SCR^B623−. Two categories were used for LSD symptoms: (a) Plants positive for LSD symptoms, or (b) plants negative for LSD symptoms. The analysis was performed across rootworm species and transgenic insecticidal technology. Therefore, the observations totaled 24 (2 insecticidal transgenic technology levels × 2 rootworm species × 2 P. ananatis acquisition levels × 3 replicates) (n = 24). This allowed us to generate the Pearson Chi-Square statistics to test the null hypothesis of independence between the events (α = 0.05) using JMP Pro 17 software [22].

3. Results

Bacterial endosymbionts and acquisition of P. ananatis isolate B623 by western and southern corn rootworms.

Adult WCR and SCR from commercial corn fields with symptoms of LSD, as well as those from the acquisition assays, were sampled and tested for endosymbiotic P. ananatis. Bacteria with different colony phenotypes were recovered on LB agar, including those with Pantoea-like round, and yellowish colonies with smooth margins. Following single-colony purification, BLAST searches identified the endosymbionts isolated from field-acquired WCR as belonging to the genera Lactococcus, Exiguobacterium, Enterobacter, and Pantoea (isolate B1188: GenBank accession: PV544963). Whereas BLAST searches of representative isolates from the field-acquired SCR were identified as belonging to the genera Pseudomonas and Pantoea (isolate B1143: GenBank and add accession PV569971). Investigations of the commercially sourced non-diapausing WCR and SCR adults for their respective endosymbiont recovered bacteria belonging to the genera Acinetobacter and Pseudomonas from the WCR, while Acinetobacter and Enterococcus were isolated from the SCR. No Pantoea species was recovered from insects commercially sourced. Three randomly selected adult WCR and SCR (commerciallysourced) were also investigated, post-acquisition assays, for the presence of P. ananatis isolate B623 that may have been acquired following ingestion during the acquisition assays. Bacterial isolates recovered from the rootworms included those that formed round and yellowish colonies with smooth margins on LB agar. BLAST searches of the sequences of representatives of the yellowish colonies, following single colony purification, found they had a hundred percent match with that of the original isolate used in the acquisition assay (GenBank accession: OP268628). No Pantoea species was recovered from the control rootworms, WCR^B623− or SCR^B623−. The recovery from the adult WCR^B623+ and SCR^B623+ used for the acquisition assays of bacteria with 16S rRNA identity matching those of the original P. ananatis isolate B623 used in the acquisition assays suggests successful acquisition by both adult rootworm species. Eggs that were subsequently produced in the rootworm cages (Figure 2) were similarly investigated for endosymbiont P. ananatis isolate B623. BLAST search and pairwise alignment results of the sequence of a representative Pantoea-like colony isolated from each of the WCR and SCR eggs identified them as P. ananatis, as well as having a hundred percent sequence identity to that of the original P. ananatis isolate B623 (GenBank accession: OP268628). BLAST searches of the sequences of other bacterial isolates recovered from the eggs, additionally, identified bacteria belonging to the genus Epicoccum from the WCR, while Pseudomonas, as well as Chishuiella (isolate B1760; GenBank accession: PV544951), a recently described novel bacterial genus [23], having a hundred percent sequence match to Chishuiella sp. strain KLBC 657 (GenBank accession: MG722822.1), were identified from SCR. No Pantoea sp. was recovered from the eggs of WCR^B623− and SCR^B623−. Finally, investigation of the WCR^B623+ and SCR^B623+ first-instar larvae (Figure 2) similarly resulted in the recovery of Pantoea-like colonies on LBA. Also, BLAST search and pairwise alignment results of the sequence of a representative Pantoea-like colony isolated from each of the WCR and SCR larvae identified them as P. ananatis, as well as having a ninety-nine percent sequence identity to that of the original P. ananatis isolate B623 (GenBank accession: OP268628).

Root Infestation and LSD Disease Symptom Development

Comparisons of experimental plants, following the infestation of their roots with the rootworm larvae, showed that all three replicate plants infested with the WCR^B623+ or SCR^B623+-derived larvae were stunted compared with plants similarly infested with WCR^B623− or SCR^B623−-derived larvae (Figure 3a,b). Tasseling was also delayed by nine to thirteen days in plants infested with the WCR^B623+ and fifteen to nineteen days in the plants infested with the SCR^B623+-derived larvae compared to those infested with the WCR^B623− or SCR^B623−-derived larvae (Figure 3a,b). Furthermore, light-green, non-wavy streak symptoms developed on leaves of plants infested with the WCR^B623+ and SCR^B623+-derived larvae, but not those infested with the WCR^B623− and SCR^B623−-derived larvae (Figure 3c,d). Diagnosis of symptomatic leaf tissues resulted in the successful recovery of bacterial isolates, including P. ananatis isolate B623 (Figure 3e,f).

The contingency table analysis of the pooled qualitative data collected resulted in a Pearson Chi-square statistic of 24 (N = 24, df = 1, χ² = 24) with a p-value less than 0.0001. Additionally, the analysis yielded an R-squared value of 1. Therefore, the null hypothesis of independence between plants exposed to rootworm larvae from different acquisition treatments and the development of LSD symptoms was rejected. Since all plants, regardless of transgenic insecticidal technology, infested with WCR^B623+ or SCR^B623+-derived larvae developed LSD symptoms, there is a perfect association between these two events (R² =1). Similarly, there is a perfect association (R² = 1) between plants not developing LSD symptoms (regardless of transgenic insecticidal technology) and infestation with WCR^B623− or SCR^B623−-derived larvae (

Table 1. Contingency analysis of plants exposed to rootworm larvae derived from the acquisition bioassays and the development of the LSD symptomatology ¹.

	Plants Showing LSD Symptoms
Rootworm Larvae Used in the Transmission Bioassay	Negative	Positive	Column Total
P. ananatis B623 − larvae	Observed: 12 Expected: 6 Cell Chi-square: 6	Observed: 0 Expected: 6 Cell Chi-square: 6	12
P. ananatis B623 + larvae	Observed: 0 Expected: 6 Cell Chi-square: 6	Observed: 12 Expected: 6 Cell Chi-square: 6	12
Row total	12	12	24

¹ N = 24, df = 1, R² = 1. χ² = 2, p ˂ 0.0001.

).

4. Discussion

The rapid spread of the disease throughout the Texas Panhandle, the edge effect, but more importantly, the patchy distribution of symptoms of LSD in affected fields, an attribute of soilborne and insect vectored diseases, led us to hypothesize a possible vector role in the dissemination and transmission of the disease-causative bacterial pathogen. The recovery of P. ananatis from among the bacterial endosymbionts of natural populations of both WCR and SCR found in fields with LSD infection added credence to the vector hypothesis. The successful recovery of the original P. ananatis isolate B623 used in the acquisition assays from the adult WCR^B623+ and SCR^B623+ is consistent with the findings by Krawczyk et al. [8], which similarly demonstrated the successful acquisition of P. ananatis strain M241 by WCR when introduced to corn on which they fed. However, in the current work, we further investigated and confirmed successful transmission of the acquired bacterium to the progeny eggs and resultant first-instar larvae of the respective rootworms. Vertical transmission by P. ananatis strain M241 in WCR was not investigated in the study by Krawczyk et al. [8] and, therefore, unknown in the case of the P. ananatis M241, which causes white spot disease in corn. The current study also differs in the exposure time to P. ananatis B623 during the acquisition assay. Whereas the acquisition assay lasted for 14 days in the study by Krawczyk et al. [8], the duration was 28 days in the current study. However, neither the relationship between exposure time and vertical transmission of the acquired P. ananatis B623 bacterium, if any, nor the minimum exposure time for successful vertical transmission was investigated in this study, and therefore not known. Nevertheless, the successful acquisition and vertical transmission of the bacteria in both rootworm species is also consistent with the observation of P. ananatis in the natural population of both adult rootworms and, consequently, validates our vector hypothesis. It is also likely, and worth noting, as demonstrated by Krawczyk et al. [8], that some of the adult field populations of both rootworms in the LSD-affected corn fields that tested positive for P. ananatis acquired the bacterium while feeding, as adults, on infected corn stands. Furthermore, neither the evolutionary timeline since the development of the original association with the rootworms, nor the epidemiological significance of either or both in the establishment of LSD in corn or sorghum, is currently known. Additionally, the specific life stage, adult or larvae, of rootworms that is most critical or essential in the natural acquisition of the pathogen in the environment was not investigated in this study and is not currently known. Neither was the potential of the bacteria to survive and persist in diapausing rootworm eggs investigated. The genetic and evolutionary potential for the development of the association between P. ananatis and the rootworms, however, likely already existed among members of the genus Pantoea. For instance, SCR was previously reported to vector Pantoea stewartii, the corn Stewart’s wilt pathogen [14], as well as a bacterium belonging to a closely related genus, Erwinia tracheiphila [15]. Similarly, WCR was previously reported to vector the P. ananatis, which causes the white spot disease of corn [8]. However, it is not known if the establishment of the association preceded the establishment of the LSD in plants or occurred afterwards.

Our findings of the development of LSD symptoms on plants infested with WCR^B623+ or SCR^B623+, but not those infested with WCR^B623− and SCR^B623−, indicate that the events are significantly linked (R² = 1, χ² = 2, p < 0.0001). Furthermore, the successful recovery of the original P. ananatis isolate B623 from symptomatic tissues indicated that neither the Bt nor RNAi technologies in the evaluated corn hybrid were able to prevent transmission of the pathogen to healthy corn plants. This, in turn, suggests that transmission from the infected larvae likely occurred before the ingestion of the equivalent effective lethal dose of either technology from the respective corn hybrid. Based on the mode of action of Bt and RNAi anti-rootworm technologies, this might have occurred in as little as one or two days after herbivory on transgenic tissues [24,25]. This finding might pose significant challenges in disease and pest management in the Texas Panhandle. Although SCR is not considered a pest in corn in the Texas Panhandle, it can, however, pose a threat to both corn and sorghum in South Texas. Given our findings that SCR can contribute as a vector in the epidemiology of LSD, its statewide pest status, particularly in corn and potentially in sorghum, could rise exponentially. Further research is needed to better understand the contribution of the rootworm vector pathway for the bacterial pathogen in the spread, severity, and economic impact outcomes of LSD disease in affected crops. Elucidation of the role of the polyphagous adult SCR, in particular, in the spread of the disease following acquisition of the pathogen is also needed to better understand their contribution to disease severity outcomes. This will contribute to the development of an integrated and adaptive LSD and rootworm management approach in the Texas Panhandle.

5. Conclusions

The findings from this work provide evidence for a vector role in the transmission of the LSD disease bacterial pathogen, involving WCR and SCR. While the study did not investigate the rootworm life stage that is most critical in the natural acquisition of the bacteria from the environment, the findings that the bacteria can be transmitted vertically to their respective first-instar larvae have important implications for the development of an effective integrated strategy for the management of the disease/pest complex in the region.