The etiology of the ARD complex is challenging to elucidate, as unlike many other soilborne diseases, numerous causal agents have been isolated from affected sites in different parts of the world. ARD has been reported by many as a complex of different types of microbial pathogens and plant parasitic nematodes which can differ in their relative abundances between sites within a geographic region (Kelderer et al. 2012; Mazzola 1998; Mazzola and Manici 2012; Tewoldemedhin et al. 2011c; Tewoldemedhin et al. 2011b). Some of the previous replant disease studies consider soil fertility and nutrient availability as major contributor to the disease (Liu et al. 2009). But other studies have negated this hypothesis as fertilization and an increase in soil nutrients were not able to eliminate ARD (Gongshuai et al. 2018; Manici et al. 2003). We measured several soil fertility parameters such as organic matter, available P, K and several other essential nutrients at the six ARD sites but did not find any correlation with the severity of ARD. However, I would like to mention that there were no "Low" disease severity sites identified in our study, only sites with "Moderate" or "Severe" disease potential were identified. These results are in agreement with study carried out in Bohai Gulf China by Gongshuai et al. (2018) as they also did not find any direct correlation between ARD severity and soil nutrient composition. Ad hoc field observations in the NS industry suggest that summer drought stress can aggravate the severity of the disease. Similar observations have been made in New York State (Rosenberger, 2023 personal communication). However, we did not find a correlation between the severity of ARD and the physical parameters of the six ARD soils that are linked with potential moisture stress such as soil texture, drainage class, and plant available moisture (Tables 2). Our study provides evidence that ARD in Annapolis Valley is of biological origin as apple seedling growth in pasteurized soil was markedly improved when compared with non-pasteurized soil in a greenhouse bioassay, a finding that is consistent with previous studies reporting ARD as being of a biological nature (Gongshuai et al. 2018; Li et al. 2016; Tewoldemedhin et al. 2011b).
Soil microbial communities and soil biodiversity play a crucial role in soil ecosystem functions and plant health by decomposition of dead organic materials, nutrient recycling, nutrient availability to plants and suppression of soilborne disease causing pathogens and inducing plant disease resistance (Barrios 2007; Liu et al. 2014; Xu et al. 2012). We detected considerable variation in bacterial and fungal community composition and structure across ARD orchard sites. More than 50% variation was observed in bacterial and fungal community composition across ARD orchard study sites. Also, 3 bacterial, and 3 fungal classes were differentially represented at different ARD orchards sites. We did not observe any correlation between severity of ARD sites and fungal or bacterial alpha-diversity across Nova Scotia orchards.
The soils of these six orchards exhibited a remarkably stable core microbiome. The Ascomycota, Mortierellomycota and Basidiomycota were mainly identified phyla at the ARD sites that account for 95% of the total ITS2 reads. Ascomycota was revealed as the most dominant phylum present in ARD sites tested in this study. This finding is similar to previous studies that also reported Ascomycota as the most dominant phylum in ARD sites (Franke-Whittle et al. 2015; Gongshuai et al. 2018). Mortierellomycetes exhibited the greatest relative abundance in the ITS2 microbiome. Mortierellacea was the most dominant family representing 36% of the total ITS2 reads at the ARD sites in this study. The role of Mortierella sp., in ARD is not very well established. Some studies have reported these as causal agents of replant disease without isolating the organism or assessing whether or not they incite the disease (Mazzola and Manici 2012; Westcott III et al. 1987). Species of Mortierella have been reported as saprophytes in soil and are involved in decomposition of organic matter. Abundance of Mortierella spp., has been reported in healthy banana soil (Xue et al. 2015). Other studies indicated that Mortierella sp., have a mutualistic relationship with apple and have been associated negatively with the severity of ARD (Gongshuai et al. 2018) and positively related with plant growth (Franke-Whittle et al. 2015).
Fusarium oxysporum and Fusarium solani were the most relatively abundant core ASV in this study. Fusarium is a large genus that contains saprophytes, endophytes and plant and animal pathogens. In soil Fusarium are often associated with plant debris and most species are saprophytic and relatively abundant in the soil microbial community. F. solani and F. oxysporum were the most frequently isolated species followed by Cylindrocarpon spp. binucleate Rhizoctonia sp., and Fusarium spp. from apple replant orchards soil in Italy (Kelderer et al. 2012). No pathogenicity assays were performed for Fusarium spp. in their study as Fusarium were considered to be non-pathogenic on apple (Kelderer et al. 2012). Several species of the genus Fusarium also produce bioactive secondary metabolites that mediate positive interaction with host plants (Bacon and Yates 2006; Shalapy and Kang 2022). Both F. solani and F. oxysporum are considered as endophytes, as they are usually isolated from asymptomatic root tissue with high abundance not only from apple tree but several other crops (Macia-Vicente et al. 2008; Manici et al. 2003; Manici and Caputo 2010; Mazzola 1997; Mazzola 1998; Tewoldemedhin et al. 2011c). However more than 20 species of the genus Fusarium are pathogens of higher plants causing root rot, vascular wilt and storage rot (Shin et al. 2016). Fusarium is often isolated from diseased apple tree roots but most isolates representing several species did not prove to be pathogenic on apple (Dullahide et al. 1994; Mazzola 1998). Tewoldemedhin et al. (2011c) frequently isolated Fusarium from all orchards in the study, but most proved to be non-pathogenic towards apple seedlings and only two of the isolates (F. avenaceum and F. solani) were only weakly virulent on apple seedlings. Previous studies have shown that F. solani was either non-pathogenic, or had low virulence towards apple seedlings (Manici et al. 2003; Mazzola 1998). We also detected several other fungal genera in this study such as Ilyonectria, Nectria and Nectriaceae that fall into the group that were traditionally called Cylindrocarpon-like fungi (Chaverri et al. 2011). These genera are reportedly negatively involved in apple growth (Braun 1995; Franke-Whittle et al. 2015; Tewoldemedhin et al. 2011a). Our findings are in agreement with these previous studies and support the conventional work of Braun (1995) in identifying Cylindrocarpon as a causal agent of ARD.
Oomycete pathogens such as Pythium and Phytophtora species have been frequently isolated from ARD soil and apple roots with varying frequency or dominance from site to site (Kelderer et al. 2012; Mazzola and Manici 2012; Tewoldemedhin et al. 2011c). Pythium species were isolated as one of the ARD causal pathogens in the transnational European study of soils from Austria, Germany and Italy (Manici et al. 2003). Pythium, Rhizoctonia and Phytophtora were also reported as pathogens that play a direct role ARD in Washington (Mazzola 1999; Mazzola et al. 2002; Shin et al. 2016). In this study, P. attrantheridium (G. attrantheridium), P. monospermum and P. ultimum (G. ultimum) were the most relatively abundant oomycetal taxa. In a previous study, Pythium irregulare (G. ultimum) and Cylindrocarpon lucidum were identified as causal pathogens of ARD in five old orchards soils of the Annapolis Valley of Nova Scotia (Braun 1991; Braun 1995).
Several bacterial families containing potential plant growth promotion taxa were part of the core microbiome. Some members of the family Chitinophagaceae, which demonstrated high relative abundance in orchard soils have an ability to produce indole-3-acetic acid, solubilize phosphate, and possess ACC deaminase activity. Each of these attributes may function to promote plant growth (Madhaiyan et al. 2015). The Sphingomonadaceae include genera with plant growth-promoting activities. Some of these genera produce phytohormones salicylic acid, gibberellins, indole-3-acetic acid and abscisic acid (Yang et al. 2014) and induce host-plant systemic resistance (Chapelle et al. 2016; Hahm et al. 2012). The presence of Solibacteraceae in the plant rhizosphere was linked to plant resistance to Fusarium pathogens (Mendes et al. 2018) and Actinobacteria and Pseudonocardiaceae exhibit antimicrobial ability against some bacteria and fungi (Chaouch 2018).
Microorganisms that play an important role in the turnover of organic plant material and soil fertility were also a part of core microbiome. The family of Xanthobacteraceae, contains potential nitrogen fixers, and degraders of alkenes, halogenated aliphatic and aromatic compounds, terpenes, thiophenes, or polyaromatic compounds (Oren 2014). Members of family Nitrosomonadaceae contains species involved in nitrification, sulfur cycling and plant growth promotion (Prosser et al. 2014). Many strains belonging to family Micromonosporaceae can degrade chitin, cellulose, lignin, and pectin (Trujillo et al. 2014). In addition to plant beneficial microorganisms, potential phytopathogens were found in the core microbiome. The Xanthomonadaceae were highly abundant in these soils. Several species of the genus Lysobacter; that belong to this family have been shown to effectively control fungal and oomycete plant pathogens (Hayward et al. 2010; Kobayashi and Yuen 2007{Hayward, 2010 #155) and cyst nematodes (Yuen et al. 2018).
Of the six genera of plant-parasitic nematodes found in the orchard soils, only root-lesion and dagger nematodes are known to be pests of apple. Our analyses did not identify the species of root-lesion nematodes in each sample, but prior research has confirmed the widespread occurrence of P. penetrans in Nova Scotia orchards, including one of the orchards (KEN) sampled in this study (Forge et al. 2019). Root-lesion nematodes are known to cause economically significant damage to apple on their own (Ark and Thomas 1936; Bélair et al. 2019; King 2022), and population densities of 30 to 100 P. penetrans / 100 cm3 soil have been proposed as approximate damage thresholds (e.g., http://www.omafra.gov.on.ca/IPM/english/apples/diseases-and-disorders/nematodes.html; https://pnwhandbooks.org/plantdisease/host-disease/apple-malus-spp-nematode-root-lesion) for apple replant. However, a recent field microplot study demonstrated apple growth reduction with an at-planting soil population density of 5.4 P. penetrans/ 100 cm3 soil (King 2022), indicating that the Pratylenchus populations observed in these Nova Scotia orchard soils would likely have measurable effects on growth of apple. As migratory endoparasites of root cortical cells, P. penetrans cause cortical necrosis of fine feeder roots, making them vulnerable to infection by opportunistic fungal pathogens, suggesting a synergistic relationship with fungal pathogens and increasing the severity of the broader replant disease complex (Mazzola and Manici 2012).
The threshold for measurable dagger nematode damage to apple has been proposed to be 50 to 100 Xiphinema/ 100 cm3 soil (e.g. Nematode | Intermountain Fruit | USU; Nematodes - Ontario AppleIPM (gov.on.ca), and we speculate that they would have affected tree growth in the three soils with population densities of 40, 149 and 172 Xiphinema/ 100 cm3. Xiphinema americanum is also a vector of tomato ringspot virus which can be a problem in apple orchards.
Pin nematodes are known to parasitize apple but only cause damage at much greater population densities than those found in these orchard soils (e.g. >500 Paratylenchus/ 100 cm3 soil). Ring nematodes, particularly the species Mesocriconema xenoplax, are known to be economically important parasites of Prunus fruit trees species (Ferris et al. 2004), but they are not often reported from apple orchards at high population densities, and there are no recorded controlled-inoculation studies of their host-parasite relationship with apple. No species of spiral nematodes have been demonstrated to be pests of apple and, similarly, no species of root-knot nematode known to exist in Canada that parasitize apple. We speculate that these nematodes are maintained in orchard soils via feeding on grasses and weeds in orchard alleys and are not of significance to apple.