Highbush Blueberry (Vaccinium corymbosum L.) and large-fruited or American cranberry (V. macrocarpon Ait.) are long-lived woody perennials that are native to North America. Cultivars of both species are clonally propagated. Blueberry is propagated from hardwood or softwood cuttings or in vitro culture and rooted prior to field planting (Mainland, 1966). Cranberry cuttings can be pressed directly into field soil or rooted prior to transplanting into the field (Eck, 1990). Establishment time can vary depending on planting location, but in the northeastern U.S., it typically takes blueberry fields 4-7 years to reach maturity. New plantings of cranberry similarly take about 4-5 years to reach full production. Once established, plantings may remain productive for many years. However, the yield of mature plantings of blueberry and cranberry sometimes declines over time. In areas where decline is prevalent, and replanting does not solve the problem, the primary cause is likely to be soil-related.

It is well established that long-term monoculture can reduce soil health (Ketcheson, 1980; Porter et al., 1997; Garside et al., 2001; Bennett et al., 2012). This decline of soil health can be complex and includes, but is not limited to, a buildup of salts and/or other toxic materials, reduction in organic matter, and a buildup of detrimental soil organisms (Tewoldemedhin et al., 2011; Mazzola and Manici, 2012). Poor performance of replanted blueberry fields, termed Blueberry replant disease (BRD) in the southeastern U.S., was found to be primarily due to pathogenic nematodes (Jagdale et al., 2013), while cranberry decline in Wisconsin was associated with Phytophthora spp. (Jeffers, 1988). In contrast, blueberry and cranberry decline in New Jersey, does not seem to be associated with either Phytophthora spp. or nematodes, although in some replanted blueberry fields where experimental nematicides were tested, plant growth improved (Oudemans, unpublished). Thus, it is likely that the cause(s) of decline in either crop varies by locality and may be due to complex community interactions, rather than a single pathogenic organism.

Blueberry and cranberry naturally grow in well-drained, sandy acidic (pH 4.0-5.5) soils that tend to be low in nutrients and with low to moderate (2-7 percent) organic matter (Eck and Childers, 1966; Eck, 1988; Eck, 1990, Pavlis et al., unpublished). Since the soils in which these crops grow is not typical, as compared to the soils in which most crops are grown, the assemblage of microorganisms in these soils is likely to be unique. While we expect to find a range of acidophilic organisms, Fierer (2017) suggested that abiotic stressors, including acidic conditions, can limit microbial survival and growth. He further notes that soil bacterial communities and abundances of specific taxa can be predicted from site characteristics which include soil pH (Fierer, 2017).

In addition to soil type, the plants growing in the soil have a profound effect on the rhizosphere microbiome (Costa et al., 2006; Berendsen et al., 2012; Marschner, 2012; Chaparro et al., 2014). We expected that because highbush blueberry and cranberry are in the same genus (Vaccinium) and grow naturally in similar soils and environments, that there would be significant commonalities in the rhizosphere communities associated with these crops. However, plant habit (1-2 m high shrub for blueberry; low-growing 0.2 -0.6 m high vines for cranberry) and management practices differ between these crops and these differences might drive some distinctions in the rhizosphere communities of these crops.

Management practices differ greatly between blueberry and cranberry. Highbush blueberry in the northeastern U.S. is still grown primarily in clean culture, i.e. all competing vegetation is controlled by tilling and herbicide use (Ballinger, 1966). The plants are pruned to remove old wood and to encourage the growth of 2-3 year old productive shoots. The plants are deciduous and go dormant for the winter. In contrast, cranberry is grown in solid stands of vines in beds of varying size with little or no exposed soil. Weeds are pulled by hand or controlled with herbicides to form a near-monoculture of cranberry vines. Cranberry plants retain most of their leaves at the end of the growing season, but still go dormant for the winter.

In this study, we sought to determine and compare the rhizosphere microbiome (bacteria and fungi) of cultivated cranberry and blueberry fields in New Jersey as a prerequisite to exploring the cause(s) of decline. While we predicted commonalities among the microbial communities based on similarity of soil type and plant genus, management practices and plant growth habit were expected to cause a divergence in at least some aspects of the microbial communities.

The fungi were remarkably similar at the phylum level, as there were no notable omissions between the rhizosphere microbiome of blueberry or cranberry. They only differed in the hierarchical abundance within phyla groups (Figure 1A), and not significantly so. Although blueberry had a significantly higher alpha diversity compared to cranberry, potentially due to distinguishing agricultural practices and possibly plant growth habit. Cranberry is typically cultivated in wetlands and this may explain why the chytridiomycota, a phylum common in aquatic environments (Barr, 1990; Gleason et al., 2008), is prevalent in the cranberry soils. In fact, the chytrid Synchytrium vaccinii is a known pathogen of cranberry and requires free water to spread (Stretch and Carris, 2017). However, fungi from this phylum were not exclusive to cranberry as they were also found in both the New Jersey (N.J.) and the Nova Scotia (N.S.) blueberry soils (Figure 1A). These findings parallel other reports of Vaccinium spp. soils, especially the identification of ascomycota and basidiomycota as major composition groups, though not with absolute ranking similarities (Li et al., 2020; Morvan et al., 2020; Zhou et al., 2022).

Many fungal genera were classified that have not been previously recognized as inhabiting the soils associated with Vaccinium spp. in the northeastern U.S. (Figures S1, S2). Detection of unique genera is not surprising considering; 1) that these methods are more sensitive than traditional isolation and culturing methods and 2) attention is typically directed toward organisms that are pathogenic or beneficial to the crop in question, thus, neutral organisms and those found in low abundance are often not characterized and/or reported. We classified the fungal genera into guilds where possible, including pathotrophs (plant, fungal, and animal), saprotrophs, symbionts, epiphytes, and endophytes. While most of the detected organisms were saprotrophs, as would be expected to colonize organic soil matter, several plant pathogens were also identified at abundances of 0.5% or greater. The presence of pathogens in the soil, even at low levels, may signal a potential disease problem since these pathogens may serve as a reservoir for an outbreak that could express under disease-conducive conditions.

Generally, of the classifiable fungal genera, blueberry contained a higher pathogen load. For example, Coniochaeta spp. are known to be pathogens of woody perennials and thus is a potential pathogen of these crops (Damm et al., 2010). Athelia rolfsii causes Southern blight of solanaceous crops, beans and many other plants, but has not been reported as a pathogen of Vaccinium spp. (Aycock, 1961). Alternaria spp. and Cladosporium spp. are known to be pathogens of blueberry and cranberry (Polashock et al., 2017). Overall, N.J. blueberry contained a higher percent abundance of the identifiable potential pathogens Coniochaeta, Fusarium, Mollisia, and Phoma compared to cranberry, with several samples harboring statistical outliers of pathogen abundance far above average (Figure 4, represented as circles). In addition, Blumeria, a species of which causes powdery mildew on grasses, was uniquely identified in N.S. blueberries (Figure 6). N.J. cranberry however, had a comparatively low pathogen load, and low pathogen diversity (Figures 3, 4, 6).

Several symbionts were also identified in the rhizosphere microbiome of N.J. blueberry and N.J. cranberry, including ericoid, ecto, and arbuscular mycorrhizae. These may represent important, but previously unknown, Vaccinium spp.- associated mycorrhizae. N.J. cranberry had a particularly high average abundance of Rhizoscyphus (note that Rhizoscyphus ericae = Hymenoscyphus ericae, but our data generally are not able to reach the species level and both genera are still listed in the databases used- hence both are used in this paper), compared to N.J. blueberry samples (Figure 3). Interestingly, it has been reported that mycorrhizae that proliferate in response to crop monoculture, may in fact be detrimental to that crop (Johnson et al., 1992). While endophytes and epiphytes were also detected, their importance in blueberry and cranberry production is not currently known. Similar to southern highbush blueberry, neither N.J. blueberry or N.J. cranberry carried a particularly high load of Oidiodendron, though cranberry carried significantly more than blueberry (Li et al., 2020 and Figure 5).

We also compared known symbionts and pathogens with another small fruit crop grown in the northeast, grape. Similar to blueberry, grape exhibited very low levels of both Oidiodendron sp. and Hymenoscyphus sp., both of which are known to be ericoid mycorhizzae (Figure 5A), though no evidence of Russula sp. was found (Figure 5C). The analysis of pathogens showed that grape and blueberry show a similarly high abundance of Fusarium sp. (Figure 5B), which is shown to be a potential cause of decline in grape (Highet and Nair, 1995; Bustamante et al., 2022).

When compared to findings in southern highbush, as described in Li et al. (2020), Hyaloscyphaceae, Leotiaceae, Pezoloma, and Hyaloscypha were not found in meaningful abundance in N.J. blueberry and N.J. cranberry.

Bacterial composition was similar between N.J. blueberry, N.J. cranberry, and N.S. blueberry. One of the top phyla however, chloroflexi, was found to differ significantly in abundance between blueberry and cranberry soils. Similarity in composition continued at the genus level, where little difference was found between N.J. blueberry and N.J. cranberry at abundances greater than 1.0%. Both N.J. blueberry and N.J. cranberry were also found to have similar bacterial alpha diversity indexes. As compared to southern highbush blueberry (Li et al., 2020), both N.J. blueberry and N.J. cranberry contained high levels of proteobacteria, acidobacteria, and actinobacteria making up the majority of bacterial phyla. This trend followed other studies that identified similarly high abundance of proteobacteria, acidobacteria, and actinobacteria in Vaccinium soils (Li et al., 2020; Morvan et al., 2020; Zhou et al., 2022). However, both N.J. blueberry and N.J. cranberry contained a heavier load of Firmicutes than southern highbush blueberry.

Only a small number of diseases of blueberry and cranberry are known to be caused by bacteria, with some of the agents being; Pseudomonas syringae, Xylella fastidiosa, Burkholderia andropogonis, Agrobacterium spp., Rhizobium spp. and Candidatus spp. (which cause phytoplasma diseases) (Polashock et al., 2017). At the genus level, several of these pathogens were identified at similar abundance in N.J. blueberry and N.J. cranberry rhizospheres including Pseudomonas, Burkholderia, Rhizobium, and Candidatus, although generally at low average abundances (<0.5%) (Figures S3, S4).

Rhizophere microbiome diversity is thought to be an important indicator of soil health (Berendsen et al., 2012; Wallenstein, 2017; Schloter et al., 2018), and comparison in diversity can help to elucidate potential pathogenic influences, nutritional weaknesses, or microbial niches that have developed in differing soil samples. To help in visualizing these complex communities, we generated representations of microbial diversity for the largest and most diverse bacterial and fungal phyla (Figures 7, 8). The Proteobacteria exhibited a high level of diversity, as indicated by the relatively high number of large hubs, of various intermediate colors, and a high level of connectivity between nodes. There do remain, however, a few nodes that appear to be dominated in either the cranberry or blueberry groups, suggesting that niches have developed for particular taxa, or that the ecosystem does not support the other associated organisms. Conversely in Firmicutes, there are few noteworthy classes that are attached to the parent phylum. Though the phylum Firmicutes may not be as large as that of Proteobacteria, of the two largest classes represented here, Firmicutes appears to be more dominated by the cranberry group. This potentially indicates that there is a lack of diversity of Firmicutes in blueberry.

Sequencing methods and bioinformatics tools are evolving rapidly and application of this technology allows us to gain insight into the rhizosphere microbiome, a key component of the phytobiome that impacts plant health. The OTUs generated from these samples were used to identify primarily fungi and bacteria that comprise the microbial communities that are associated with the rhizosphere of blueberries and cranberries in New Jersey. While we found similar communities between blueberry and cranberry in New Jersey, we noted differences between our data and that of the lowbush blueberry rhizosphere in Nova Scotia. Although lowbush blueberry (Vaccinium angustifolium) is in the same genus, the environmental conditions, management practices and soil characteristics are very different between the two locations (N.J. and Nova Scotia, Canada). Where similarity may still occur, is in characterizing the factors associated with poor plant performance as many of the diseases that are known to affect these crops overlap (Polashock et al., 2017). Using these methods, allows the detection and subsequent targeting of specific organisms using chemical control and/or modification of cultural practices. At the same time, enhancement of beneficial organisms, such as ericoid mycorrhizae, can also be monitored.

Many years ago, Waksman (Waksman, 1918) stated “The cranberry soils are so distinctly different from ordinary soils that it was thought for a long time that no very large number of bacteria can exist in them and that the microbial population consists predominantly of molds”. Though the uniqueness of the soils in which cranberries (and blueberries) holds true, we have shown that there are many phyla of bacteria and fungi that are adapted to these crops and the environments in which they grow. As we further characterize this myriad of organisms, we ultimately hope to characterize the microorganism(s) specifically associated with crop decline.

The data presented in the study are deposited in the NCBI Sequence Read Archive under BioProject ID: PRJNA956268

The project was conceived and directed by JP and PO. PO directed field collections and JP directed all laboratory work. JK and LE performed various data analyses and created the figures. JP and JK wrote the manuscript draft with contributions and editing by PO and LE. All authors contributed to the article and approved the submitted version.