Honeydew Microbes Shape Plant–Herbivore Interactions via Defense Signaling

Hemipteran insects feed on phloem sap and excrete substantial amounts of honeydew that accumulate on plant leaves. Rich in carbohydrates and microbes, honeydew is known to mediate ecological interactions between insect pests and their natural enemies. However, the mechanisms and ecological consequences of honeydew-associated microbes in plant-herbivore interactions have not been fully elucidated. In this study, we investigated the impact of honeydew deposition by the mealybug <i>Phenacoccus solenopsis</i> on herbivore fitness and the underlying mechanisms. Application of crude honeydew to cotton plants reduced weight gain in both <i>P. solenopsis</i> and <i>Helicoverpa armigera</i> and inhibited phloem ingestion by <i>P. solenopsis</i>. While sterilized honeydew also suppressed the growth of <i>H. armigera</i>, it did not affect <i>P. solenopsis</i>. Both crude and sterilized honeydew induced jasmonic acid (JA)- and salicylic acid (SA)-dependent defense responses and promoted SA accumulation in cotton. To evaluate the contribution of honeydew-associated microbes, we isolated and identified three bacterial genera, <i>Acinetobacter</i>, <i>Bacillus</i>, and <i>Staphylococcus</i>, from honeydew, using 16S rRNA sequencing. Exogenous application of <i>Acinetobacter</i> or <i>Bacillus</i> to cotton leaves suppressed weight gain in both herbivore species and reduced phloem ingestion by <i>P. solenopsis</i>. Additionally, each bacterial isolate activated the expression of JA- and SA-responsive genes and enhanced the accumulation of both hormones. These results demonstrate that honeydew-associated microbes play a key role in plant-herbivore interactions by activating plant defenses. Our study reveals that hemipteran honeydew microbes can function as exogenous elicitors of plant resistance against herbivorous insects.