Many outbreaks of human pathogenic bacteria, such as E. coli, are associated with Romaine lettuce. For example, during the mid-2018 outbreak, 210 people from 36 states were infected with E. coli O157:H7, 96 were hospitalized, and 5 died. The lettuce surface, like that of all plant leaves, is home to innumerable microbes (fungal and bacterial). However, because lettuce leaves are nearly always consumed raw rather than cooked, the microbes on the lettuce phylloplane are often ingested by the consumer. Despite this, our knowledge about natural bacterial and fungal species associated with the phylloplane of Romaine lettuce is very limited, and yet this knowledge is necessary for understanding how this naturally occurring flora interacts with introduced human pathogens on the phylloplane. Thus, characterizing these microbial communities is crucial from a public health point of view. In this project we use an integrative approach to document and characterize the fungal microbiome of Romaine lettuce. We bought 63 lettuce heads in grocery stores in Illinois, Indiana, Ohio, and Virginia. We plated homogenized samples on corn-meal agar and isolated individual cultures using axenic techniques. These cultures were DNA barcoded with the internal transcribed spacer (ITS) region of the ribosomal DNA using Sanger sequencing. In total, we isolated 330 cultures and generated ITS sequences for 242 of those, representing 63 unique species of fungi on the lettuce phylloplane. Of these, 9 are undescribed species in diverse genera – Cystofilobasidium, Holtermaniella, Rhodotorula, Sampaiozyma, Sporobolomyces, and Tilletiopsis. Interestingly, the most commonly encountered species in our Romaine lettuce cultures was an undescribed red yeast that is sister to Sporobolomyces roseus (Pucciniomycotina, Sporidiobolales). Finally, next-generation sequencing was conducted on the same lettuce homogenates, resulting in 630 operational taxonomic units (or species proxies), a tenfold of what we found with conventional Sanger sequencing. We compared abundances at different taxonomic levels (classes, orders, genera) between organic, non-organic, and hydroponic treatments. The fungal abundance at all levels was highest in organic lettuces, closely followed by non-organic lettuces. Fungal abundance in hydroponic samples was almost non-existent, comparatively. Our results indicate that certain groups of fungi, like yeasts, in organic samples are replaced by other groups, such as molds, in non-organic samples, likely in response to differing management regimes.

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Part of this work was presented at the 2019 Purdue University Microbiome Symposium. Click on the poster below to enlarge.