Detection and Implication of Early Agrobacterium tumefaciens Infection of Paradox Seeds and Seedlings

Paradox (Juglans hindsii × J. regia), the dominant rootstock used in California, USA walnut production, has many desirable horticultural characteristics, but is highly susceptible to crown gall. Crown gall, caused by the bacterium Agrobacterium tumefaciens, is not consistently controlled with soil fumigation. This observation, in conjunction with observations of galls occurring in above ground grafting wounds, led to the hypothesis that A. tumefaciens inoculum may be endophytic in walnut seedlings. Paradox seeds collected directly from commercial Paradox mother trees (i.e., Juglans hindsii) were free of A. tumefaciens. Sampling conducted at multiple times during seed production revealed that seed-borne A. tumefaciens populations increased as a function of contact time with the orchard floor. In greenhouse experiments, seeds inoculated with A. tumefaciens produce seedlings with galls at the crown and/or roots in the absence of artificial wounding. Endophytic populations of A. tumefaciens were also found in shoot and root tissue. These data suggest that A. tumefaciens inoculum may exist as a resident endophyte and that timely seed collection, limiting contact with the orchard floor, may be an important first step in preventing crown gall disease of walnut. INTRODUCTION Paradox (Juglans hindsii × J. regia) is the dominant rootstock used in California, USA walnut production because it is precocious, vigorous, more resistant to Phytophthora root and crown rots, and enhances walnut yields. However, Paradox is susceptible to crown gall disease caused by the soil-borne bacterium Agrobacterium tumefaciens (Ogawa and English, 1991; Ramos, 1998; Flint, 2003). Soil-borne populations of A. tumefaciens are hypothesized to be the principle source of inoculum. Consequently, the primary management strategy for crown gall is preplant soil fumigation (Ogawa and English, 1991; Agrios, 2005) which has proven to be inconsistent (Ramos, 1998; Flint, 2003). Nurserymen, growers, and University of California Extension Farm Advisors have provided numerous accounts of crown gall outbreaks in fumigated walnut orchards. Additionally, in several fumigated nursery sites, accounts exist of galls forming not only on the crown and roots, but at grafting wounds well above the soil line. Galls forming on above ground parts were correlated with gall present at the crown or roots. In combination, these observations lead to the hypothesis that, in addition to incomplete fumigation, A. tumefaciens contaminated asymptomatic Paradox seeds or seedlings, may serve as a source of inoculum. In production orchards, these infected asymptomatic seedlings may result in systemic populations of A. tumefaciens which results in above and below ground galls despite soil fumigation. These circumstances are similar to crown gall of grape where Agrobacterium vitis can establish a systemic population in asymptomatic grape vines. Symptoms manifest only where frost damage causes wounds (Agrios, 2005). If A. tumefaciens can establish asymptomatic, systemic infections, contaminated seeds and seedlings would explain the outbreaks of crown gall in seemingly inoculum free soil. For these reasons, investigations testing the hypotheses of asymptomatic and/or systemic populations in seeds/seedlings were initiated. Here we report on the preliminary investigations examining; i) the Proc. VIth Intl. Walnut Symposium Ed.: D.L. McNeil Acta Hort. 861, ISHS 2010 498 prevalence of A. tumefaciens in Paradox seeds and ii) the prevalence of systemic populations of A. tumefaciens in walnut seedlings resulting from contaminated seeds. MATERIALS AND METHODS