Differential susceptibility of spinach grown under slow- and fast-growth conditions to enteric bacterial contamination.

Spinach is grown in the Salinas valley during most months of the year. Early season spinach takes 40-50 days (slow growth) to reach harvest stage whereas spinach grown during the warmer summer months grows much more quickly, taking 30-35 days (fast growth) to reach harvest stage. Spinach grown under fast-growth conditions is subject to significant post-harvest breakage which may influence susceptibility to coliform bacterial colonization. We propose to compare three spinach varieties grown under slow- and fast-growth conditions for physiological differences that may contribute to leaf structure and stability (waxy layer, plasmalemma and cell wall thickness, number of chloroplasts, etc.) and susceptibility to E. coli O157:H7 colonization. We will also add an additional stress on the plants, that of insect damage, to see if damage combined with fast-growth conditions result in higher E. coli counts. Our goal is to document the physical changes in spinach that accompany fast-growth conditions and identify increased risks for E. coli contamination of summer-grown plants. This will be the first step in systematically testing a larger number of spinach cultivars for traits that minimize breakage and contamination risks.

Minimally processed leafy greens are cut, then washed in preparation for bagging and distribution. During this process, some leafy material is subject to breakage, which is defined as increased damage to the leaf manifested as folding, water soaking, and bruising. This phenomenon is particularly prevalent in the summer months of July, August, and September, when growing conditions are particularly favorable for rapid spinach growth. The current rationale is that the plants are growing so rapidly during June, July, and August, that leaf structure is unstable; leaf structure is weak and subject to collapse under the pressure exerted under washing conditions. However, this has not been well documented. To document this type of damage, we propose to examine the physical parameters of spinach grown under conditions that promote slow growth and those that promote fast growth to see if there are physical traits that support the contention that the spinach leaf structure is weaker or is of reduced structural integrity when grown under fast-growth conditions using transmission and scanning electron microscopy. Another complicating factor is that the increase in leaf breakage coincides with increased incidence of E. coli O157:H7 detection in spinach. Is there a connection? We further propose to compare spinach cultivar susceptibility to E. coli O157:H7 contamination and colonization when spinach is grown under fast- and slow-growth conditions using a combination of microbiological detection and fluorescent microscopy. Finally, because damage to the leaf has been shown to be critical to bacterial colonization, we will compare susceptibility of spinach cultivars grown under fast- and slow growth conditions after they have been exposed to common piercing sucking insects. These objectives address Core Safety Research Needs 1.3 Leafy Greens Growth Rate and Pathogen Survival and 1.5 Other Animal Risk Factors. If successful, this project will be the basis for screening of large numbers of spinach cultivars for physical characteristics that favor resistance to leaf breakage and bacterial colonization of the leaf surface.

Objective 1: Using light, transmission, scanning electron microscopy, compare physical parameters of spinach varieties grown under slow‐ and fast‐growing conditions. 

Objective 2: Determine susceptibility to bacterial colonization of spinach varieties grown under slow‐ and fast‐growing conditions using a fluorescently‐tagged strain of E. coli O157:H7. 

Objective 3: Compare susceptibility of spinach to E. coli colonization after insect damage using fluorescent microscopy.

1. Leaves of slow growth plants were 30‐50% thicker than those of fast‐growth plants. 

2. Both types of leaves had approximately the same number of cells per unit area, but the slow‐growth cells were larger and the cell walls were thicker. 

3. The surface of slow growth leaves were smoother than those of fast‐growth leaves 

4. Cuticular wax composition differed between fast‐ and slow‐growth leaves. 

5. Slow growth leaves were stronger than fast‐growth leaves, especially after wilting slightly. 

6. Salmonella attachment to slow‐growth leaves was higher than attachment to fast‐growth leaves, but the variation was too high to show significant differences. 

7. Similarly, when broken leaves were compared to unbroken leaves, mean number of Salmonella cells attached to the unbroken leaves was higher. 

8. Tensile strength tests showed that the slow‐growth leaves were stronger (less susceptible to tearing) than fast‐growth leaves after a period of wilting. Recommendations: We have been able to demonstrate physiological and physical differences in a single variety of spinach (Silverwhale) using growth chamber‐grown plants. These plants, while they strongly resemble field grown material, should not be considered equivalent to field‐grown spinach. A number of factors that were examined in this study suggest that spinach grown in the summer months is weaker than spring‐grown spinach and may benefit from different harvesting/processing protocols to avoid the stresses that result in ‘breakage.’ This may include management strategies designed to slow down the rapid growth during the summer months or harvesting techniques that minimize physical stresses on the leaves. The question of increased/decreased pathogen attachment to fast‐ or slow‐growth leaves or to broken vs. unbroken leaves was not adequately answered. In my opinion, this may not be possible to test in the laboratory. It is my suggestion that this question be addressed, but with closer collaboration with a leafy greens processor. NewStar sent me samples whenever requested, but it would have been better if field grown spring and summer spinach could have been processed and tested on site, rather than have the samples shipped to me. A larger scale experiment of contamination and recovery of pathogen would have been better for this particular research objective.