Fumigation with dazomet modifies soil microbiota in apple orchards affected by replant disease

Highlights

• Fumigation with dazomet increased growth and yield in ARD-affected apple orchards.

• Fumigation induced medium/long term modifications in soil microbial communities.

• A complex of potential pathogenic fungi was correlated with reduced plant growth.

• In fumigated plots, there was an increase in plant growth promoting microorganisms.

Abstract

Apple replant disease (ARD) is a disorder that affects apple trees when they are replanted in soil where the same species was previously grown. ARD has been known for a long time, but the precise cause is not yet identified. Although ARD is most probably due to a combination of abiotic and biotic factors, the fact that soil fumigation commonly prevents the symptoms, at least temporarily, supports the hypothesis that microorganisms play an important role in it. In order to find possible relations between taxa composition of soil microbial communities and plant growth in ARD-affected orchards, we compared fumigated (dazomet 99%) and untreated soils by using next-generation sequencing (NGS) technologies. Soil sampling was carried out when the difference between fumigated and untreated plots became significant in terms of shoot growth and fruit yield and specifically at the end of the second growing season. Total soil DNA was extracted and two target regions (ITS for fungi and 16S rDNA for bacteria), were pyrosequenced with Roche’s 454 Platform. Both bacterial and fungal communities differed significantly in fumigated and untreated soils of our study. Bacillus sp. (ρ = 0.64), Streptomyces sp. (ρ = 0.64), Pseudomonas sp. (ρ = 0.59), and Chaetomium sp. (ρ = 0.85) were some of the taxa positively correlated with asymptomatic apple trees. Although a cause-effect relation with ARD cannot be proven, our results confirm that, fumigation with dazomet reduces ARD symptoms, and also modifies soil microbial communities at length, in particular by increasing the presence of some beneficial microorganisms known for their action against plant pathogens.

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).