Increased populations of deleterious fluorescent pseudomonads colonizing rhizomes of leatherleaf fern (Rumohra adiantiformis) and expression of symptoms of fern distortion syndrome after application of Benlate systemic fungicide

Abstract The overall objective of the current study was to test two hypotheses: (1) application of the systemic fungicide Benlate to leatherleaf fern results in long-term increases in populations of deleterious fluorescent pseudomonads that endophytically colonize rhizomes; (2) such endophytic colonization, resulting from Benlate treatment, is associated with damage to leatherleaf fern and development of symptoms of fern distortion syndrome (FDS). Mean populations of fluorescent pseudomonads and total aerobic bacteria in the rhizosphere were significantly increased 3 months after the application of Benlate 50 WP and Benlate 50 DF as foliar sprays and drenches, compared to populations in the non-treated control. In another test, at 5 months after treatment, endophytic populations of fluorescent pseudomonads inside rhizomes of plants treated with Benlate 50 WP and Benlate 50 DF (both from DuPont) were significantly higher than populations in rhizomes of plants treated with three different generic sources of Benlate 50 WP, the active ingredient benomyl, and MBC. In the same test, treatments with the two DuPont sources of Benlate, but not the generic sources, benomyl, or MBC resulted in reduced overall growth of plants, including reductions weight of fronds, roots and rhizomes and caliper of rhizomes. Three long-term experiments examined the effect of Benlate 50 WP and Benlate 50 DF on populations of fluorescent pseudomonads and development of distortions of frond growth and other symptoms associated with FDS. In two tests at 24 months after application, treatment with both formulations of Benlate increased endophytic populations of fluorescent pseudomonads inside rhizomes, compared to controls, in both experiments, and endophytic populations inside petioles of fronds arising from rhizomes were significantly greater with both Benlate treatments in one test where this parameter was measured. Also at 24 months after application, the Benlate treatments were associated with significant increases in severity of FDS, using a previously reported rating scale and by measuring several other parameters associated with FDS. In the third long-term test, the first crop of fronds was cut 24 months after treatment. Four weeks later, the numbers of actively growing rhizomes (that had produced new fronds) were significantly reduced on Benlate-treated plants, compared to the controls. At 6 months after cutting (30 months after Benlate treatments), weights of fronds were lower on Benlate-treated plants than on controls. Characterization of fluorescent pseudomonads isolated from inside rhizomes and petioles at 24 months after treatment revealed that applications of Benlate resulted in a marked increase in pectinolytic activity. DNA sequencing of 150 strains of fluorescent pseudomonads from inside rhizomes and 60 from inside petioles at 24 months after application of Benlate indicated low matches to type strains in the ribosomal data base. Phylogenetic characterization of the strains indicated the existence of 10 clusters. Pronounced shifts in frequency of strains in the various clusters were noted with Benlate treatment: over 70% of strains from rhizomes of Benlate-treated plants belonged to cluster E3, compared to only 2% of the strains from control rhizomes, and 98% of the control strains belonged to clusters C, D2, F, and G, while none of the strains from petioles or rhizomes of Benlate-treated plants belonged to these four clusters. In summary, treatment of leatherleaf fern with Benlate 50 WP and Benlate 50 DF from DuPont led to a progression of long-term deleterious effects on leatherleaf fern that were associated with increased populations of fluorescent pseudomonads that were functionally and phylogenetically different from pseudomonads from control plants.