Spatial Distribution of Crown Gall in a Commercial Nursery of Weeping Fig

Agrobacterium larrymoorei causes tumors on weeping fig. The association between propagation and pathogen spread in mother trees and daughter branches was studied in a commercial nursery. The mother tree planting was scouted for tumors prior to and after propagation. Branches selected for propagation were tagged to track disease development. The spatial distribution of crown gall in the mother tree planting was characterized with runs, join-count, and spatial autocorrelation analyses. The association of disease in mother trees and daughter branches was characterized with cross-correlation analysis. The incidence of crown gall in the mother tree planting increased from 7% prior to propagation to 32% eight months after propagation. Of the 4193 daughter branches monitored, 3.8% developed tumors. Runs analysis indicated significant clustering of diseased mother trees. Significant cross-correlations between mother trees and daughter branches with symptoms of crown gall were detected out to a distance of two plants from the source. Although pruning shears were routinely soaked in a disinfectant in this nursery, the degree of sterilization achieved apparently was not sufficient to prevent pathogen transmission. This study suggests that alternative sanitation measures should be sought and that infected mother trees and their neighbors should be avoided for propagation. Introduction Crown gall on weeping fig (Ficus benjamina L.) has been a problem in commercial production of F. benjamina since 1991 (1). The disease is characterized by tumors on the main trunk of the plant as well as aerial tumors on branches (Figs. 1A, B). The bacterial pathogen causing crown gall of Ficus spp. is likely endemic to South Florida. Crown gall was first reported on the roots of Ficus aurea in the Florida Everglades in 1919 (8). Aerial tumors appear to be associated with pruning wounds that are made during propagation and general plant maintenance (1). The pathogen is thought to have been introduced into Florida’s commercial braided Ficus production after the freeze of 1989 when aerial tumors were found on 1-year-old trees produced from cuttings obtained from old hedges in South Florida (1). It was later discovered that bacteria isolated from aerial tumors were distinct from previously identified strains of Agrobacterium based on their unique opine metabolism and 16S rRNA sequence differences (1). In 1995, the tumorigenic Agrobacterium strain isolated from F. benjamina was identified as a new species and was named Agrobacterium larrymoorei sp. nov. (2). The braided Ficus plant that is commonly encountered in retail stores is produced by first creating air layers on branches of similar size on mother trees of mature F. benjamina (Fig. 1C). Air layering is a propagation technique used to induce root formation on a branch while it is still attached to the parent plant, and is used on plant species that are difficult to root. To create an air layer, a 4cm ring of bark and cambium layer is removed from a pencil-sized diameter branch approximately 40 cm from the distal end of a branch. Alternatively, a 3to 5-cm cut can be made at an angle into about the center of the branch, and a small sliver of wood (e.g., a piece of toothpick) is inserted into the wound to hold it open and prevent it from healing. Next, a clump of dampened sphagnum moss is wrapped around the branch at the site of the wound and aluminum foil is 26 November 2012 Plant Health Progress wrapped around the clump of moss to hold it securely in place and prevent moisture from escaping. The foil is secured to the branch either by twisting it around the branch protruding from either side of the clump of moss or with twist-ties. Once the roots have formed − about 3 to 4 weeks after creating the air layer − the daughter branches are pruned from the parent plant just below the root ball with sterilized shears and 3 to 4 similarly sized daughter branches (not necessarily originating from the same mother tree) are braided, potted in sterile potting media, and then placed in nursery beds for 3 to 4 weeks to allow the plants to fuse and heal. Wounds to the mother tree and the newly formed daughters created during the process of air layering, as well as wounds produced when braiding the daughter branches, are typical sites of tumor formation (22). Having knowledge specific to the epidemiology of crown gall on Ficus is useful when developing disease management strategies. This knowledge can provide insight into which production practices might be effectively altered to disrupt disease development. For Ficus, the extent to which crown gall is transmitted in the mother planting during the process of propagation and the extent to which crown gall is passed to the daughter plants are not well understood. The objectives of this project were to: (i) determine the effect of propagation on transmission of crown gall within the mother planting; and (ii) determine the likelihood that air-layered daughter branches originating from symptomatic mother trees result in infected daughter plants. 26 November 2012 Plant Health Progress Disease Monitoring and Data Collection The study was conducted in a commercial nursery located in St. Lucie Co., FL. The mother tree planting of F. benjamina cv. Wintergreen consisted of 16 evenly spaced rows of 78 plants and was initially surveyed for tumors by nursery personnel just prior to the beginning of propagation in 2007. This was followed by a confirmatory scouting by laboratory personnel of the author during the process of tagging branches selected for air layering (discussed below). Three periods of propagation occurred within this field which coincided with orders Fig. 1. (A) Tumors caused by Agrobacterium larrymoorei sp. nov. on the main trunk of weeping fig (Ficus benjamina L. cv. Wintergreen) as well as (B) aerial and crown tumors on daughter branches. (C) Example of air-layers (wrapped in blue foil) on weeping fig. (D) Example of air layers that were numbered and tagged with weather-resistant aluminum tags as discussed in the text. (E) Braided plants that were placed on ground-cloth-covered plots to allow the branches to fuse and heal and form a single braided plant. 26 November 2012 Plant Health Progress placed at the nursery (Table 1). Beginning with the first row of the mother tree planting and winding through the rows in a serpentine pattern, employees of the nursery inspected each tree of the planting and selected branches for air layering based on a combination of vigor, length, and circumference of the branch until the necessary number of branches was chosen to meet the order. The branches were selected from mother trees with no visible symptoms of crown gall. Subsequent periods of propagation began in the row where the last propagation event ended, except that row 7 was utilized in both the 1st and 2nd times. Within 3 days of the start of propagation, the air layers were numbered and tagged with weather-resistant aluminum tags (Al Tag, Forestry Suppliers Inc., Jackson, MS) for future tracking (Fig. 1D). After three to four weeks, air-layered branches that had formed roots, herein referred to as daughter branches, were cut from the mother tree, braided with 2 to 4 other daughter branches, planted in pots containing sterile potting media, and then placed on ground-cloth-covered plots to allow the branches to fuse and heal and form a single braided plant (Fig. 1E). This process was completed by employees of the nursery. Tumor formation was tracked on all tagged daughter branches (3 to 4 per pot) at approximate monthly intervals. With the braided plants, it was sometimes difficult to associate a tumor to a single daughter branch because some tumors formed evenly at the center of the interface where branches intertwined. In these cases, the tumor was assigned arbitrarily to a particular daughter branch, but often this was inconsequential because for any single braided plant the daughter branches were routinely pruned from the same mother plant. In 2008, a final survey of the mother tree planting was conducted by lab personnel approximately 6 to 8 months after the last propagation period concluded (Table 1). The lag time between the last propagation period and the final disease rating was to allow sufficient time for tumor development so they could be more easily identified within the dense foliage. The typical incubation period for crown gall diseases (i.e., the time between inoculation and symptom development) is 2 to 4 weeks. Table 1. Dates of the propagation activities conducted in a commercial planting of weeping fig (Ficus benjamina L. cv. Wintergreen). x Air layering and subsequent production of daughter plants was done in three distinct time periods: Period I utilized rows 1-7 of the mother tree planting; Period II utilized rows 7 to 12; and Period III utilized rows 13 to 16. Mother tree rows selected in each period of propagation typically began where propagation ended in the previous period except for row 7 which was utilized in both Periods I and II. y The nursery plot was a ground-cloth covered area where potted, braided Ficus plants were placed to be managed while they fused and healed. Distribution and Incidence of Crown Gall The distribution of mother trees with symptoms of crown gall and the number of daughter branches from each mother tree that developed tumors in their nursery plots are shown in Figure 2. A total of 1129, 1805, and 1259 daughter branches were collected and monitored for symptoms of crown gall from the first, second, and third period of propagation, respectively. The Periodx