Fumigation with dazomet modifies soil microbiota in apple orchards affected by replant disease
Introduction
Apples are an important crop, which accounts for a production of more than 80 million tons worldwide (FAO, 2013). They are commonly grown as a highly specialised monoculture in regions where the climate is particularly favourable for fruit quality. In these areas, characterised by high land value, growers are unlikely to implement crop rotation, so apple orchards are commonly replanted immediately, which quite often results in reduced yield over time. This problem has been named apple replant disease (ARD) (Ross and Crowe, 1973).
The main symptoms of ARD are a general reduction in plant growth, fruit yield and quality; plants have shortened internodes, discoloured roots, root tip necrosis and a reduction in root biomass, which can lead to plant death within the first growing season (Mazzola and Manici, 2012). Poor growth and production caused by ARD may decrease profitability by up to 50% throughout the lifespan of the orchard (van Schoor et al., 2009).
The causes of ARD are still unclear, despite the fact that research has been undertaken for decades. The most plausible hypothesis is that ARD is the result of the activity of soil pathogens/parasites, although other factors cannot be excluded (Mazzola and Manici, 2012). Fungal species belonging to the Cylindrocarpon, Rhizoctonia, Phytophthora and Pythium genera are frequently found in ARD-affected soils, but their presence and frequency can vary from soil to soil (Tewoldemedhin et al., 2011a, Tewoldemedhin et al., 2011b). The role of prokaryotes in ARD has been little investigated and opinions on their involvement in the disease are contrasting (Hoestra, 1968, Mazzola, 1998). The severity of ARD symptoms can be influenced by environmental factors, such as water stress and salinity (Redman et al., 2001), general soil fertility (Braun et al., 2010) and the presence of phytotoxic compounds (Tagliavini and Marangoni, 1992), hence drawing up a complete picture of the disease aetiology is complex.
ARD has long been studied with classic soil microbiological approaches (i.e. isolation of soil microorganisms on selective agar media and subsequent identification). However, these techniques, well suited for the detection of known pathogens, are inadequate for studying the whole soil microbial community, because only a minimal part of the soil microbial community is cultivable on laboratory media (Guo et al., 2014, van Schoor et al., 2009). The high throughput sequencing technologies allow studying microbial communities in a complex ecosystem (Daniel, 2005) and may help in better understanding ARD, by analysing in depth the entire bacterial and fungal community.
Soil disinfestation prior to replanting with pasteurisation or fumigation can partially or temporarily relieve ARD symptoms (Covey et al., 1979, Mai and Abawi, 1981), supporting the hypothesis of a microbial role in the syndrome. With fumigation, a volatile chemical compound is applied to the soil, which is then covered with plastic film to favour gas diffusion into it and avoid the dispersion of the active substance during the treatment. The treatment kills most soil-borne pests and pathogens (Eo and Park, 2014).
Dazomet (tetrahydro-3,5-dimethyl-2 H-1,3,5-thiadiazine-2-thione) is a granular fumigant that releases methyl isothiocyanate, which is often used to treat soil before apple replanting. It is effective against several pathogenic microorganisms, nematodes and weeds, and this treatment commonly results in enhanced yield in comparison to untreated soils (Otto and Winkler, 1993).
The environmental risks of synthetic chemical soil fumigants are frequently debated and no exhaustive information is available on the long term impact of dazomet on soil microbiota. Some studies on the short term effects of dazomet on soil microbial communities in microcosms a few days after application (Eo and Park, 2014, Feld et al., 2015) have shown a decrease in richness and biodiversity. However, the effects after a longer period of time (e.g. one year or more) have not yet been investigated.
The aim of this study was to compare microbial communities in fumigated and untreated ARD-affected soils in order to verify whether the presence of some groups of soil microorganisms could be associated with ARD. Soil sampling was performed in an apple-growing area in northern Italy and the differences in composition and abundance in the soil microbiome were assessed when ARD symptoms became evident (at the end of the second growing season, 19 months after replanting).
Section snippets
Study site and composite soil sampling
The study site was located in northern Italy (Trentino-South Tyrol region) in the alluvial plains of the Adige River, an area of intensive apple production (Municipality of Ora, 46.0 N, 11.3 E). The soil at the site originated on recent alluvial deposits and was classified as Typic Fluvaquent, coarse silty, mixed, mesic (Soil Survey Staff, 2010). The area was selected because it is homogeneous in terms of climate and soil type and was continuously cultivated with apple trees for several
Soil properties, soil cultivable microorganisms count and ARD severity at the end of the second growing season
The soil texture was silt-loam in all the plots and no difference in the chemical composition in fumigated and untreated plots was found (Table 1; t-test, p > 0.05 for each chemical parameter).
Regarding soil cultivable microorganisms, the number of CFUs did not vary significantly according to the soil treatment (t-test, p > 0.05). In the fumigated plots, we counted 4.3 106 ± 2.7 106 and 7.5 104 ± 5.0 104 CFUs g−1 dry soil (average ± standard deviation), for bacteria and fungi, respectively. In the
Overall differences in microbial communities after 19 months from fumigation
As expected, microbiological techniques did not allow identifying significant differences in bacterial or fungal CFUs between fumigated and untreated soils. Despite the risk of PCR biases, NGS technology is, to date, one of the best approaches to have a comprehensive view of the microbial community, because cultivation techniques can capture less than 1% of the microbial biodiversity of soil and they were already found insufficient to fully describe the microbial complexity in several previous
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