Replant disease describes a phenomenon of disturbed physiological and morphological reactions of plants after replanting crop species at sites previously used for similar crop cultures [1
]. Replant disease has been reported for several horticultural crops, including apples, peaches and cherries in nurseries and orchards all over the world [2
]. On apple trees, symptoms of replant disease include damaged root systems; stunted growth above and below ground; and reduced fruit yields [1
]. While the direct cause of the soil-borne replant disease has not been revealed, it has been attributed to a plethora of potential biotic and also abiotic factors. Biotic factors are generally believed to be the predominate causal agents of replant disorders, since replant soils treated with soil fumigation, soil pasteurisation and soil sterilisation have shown restored regular plant growth [5
]. Convergence has evolved around genera of oomycetes (Pythium
); actinomycetes or bacteria (Bacillus
); and multiple fungal species (e.g., Cylindrocarpon
-like fungi, Rhizoctonia
) that appear to contribute to the complex disease [9
]. However, a definite relation between sequence data and replant disease in the microbiome of replant soils has not been shown yet [12
Abiotic factors, on the other hand, are understood as influences regulating the extent of the symptomatic effect of replant on tree vigour, rather than a primary cause [1
]. Abiotic factors include water logging, soil pH and (micro)nutrient deficiencies [1
]. Replant-sensitivity of apple trees has been found to differ by soil type [13
] and soil texture [11
]. Tewoldemedhin et al. (2011), for example, grouped the status of the apple replant disease (ARD) by growth response in non-treated versus pasteurised replant soil: low ARD—status on soils of clay and loamy texture; moderate ARD—status on soils of loamy texture; and severe ARD—status on soils of sandy texture [11
Replant-related suppression of apple tree growth performance has been found individually pronounced between apple understocks and trees, respectively [17
]. The suppression results in an uneven growth by heterogeneous distribution of more or less replant, symptomatic apple plants across replanted apple orchards. The suppression of tree vigour in apple trees, and the consequential lack of a development of best-performing trees across the orchard, leads to decreased profitability of yields that can add up to 50% throughout the life cycle of replanted orchards [19
By meta-analysis, Nicola et al. (2018) showed that the soil microbial community significantly differs under replant conditions [12
]. However, shifted microbial communities show relatively small overlaps of microbial constituents between geographically distantly located replant sites, indicating site-specific replant effects on the soil microbiome [12
]. In field studies, ARD-symptomatic and non-symptomatic trees have been associated with shifts in the density of several soil fungi, including the class Dothideomycetes,
and more specifically in the order Pleosporales
]. The genetically determined Alternaria
-group (Ag) (order Pleosporales
, family Pleosporaceae
) has been identified as a replant-sensitive soil fungal population which responds to replanting by abundance [19
]. The proportion of Ag on the total soil fungal population was found to be 2% in replant soil; this was found to be 10-fold greater compared to no-replant soil. Such slight shifts in the Ag population can reflect larger shifts in the soil fungal community (beyond Ag) and thus be indicative for shifts in the distribution of sieve-size fractions and aggregate stabilities.
Most microbial studies indicating replant-related or even causal agent(s) of replant disorder have focussed on homogenized soil samples. Soil microbial interactions, however, occur in habitats much smaller than those generally captured in homogenized soil cores [21
]. Microbial community composition is strongly mediated by soil structure [22
]. The general heterogeneity of the soil structure supports a high diversity of microhabitats with different physico-chemical gradients and discontinuous environmental conditions [24
], even when the overall environment of the soil is constant [23
]. Specific microbial taxa have habitat preferences that are linked to the morphological, chemical and physical properties of the interior and exterior interfaces of soil aggregates [26
]. In general, the proportion of fungi within soil aggregates varies within aggregate size, as a greater proportion of fungi have been associated with macro-aggregates (>250 µm), whereas bacteria were mainly associated with micro-aggregates (≤250 µm) [21
]. The microbial community was also found to vary within and among aggregate fractions of the same soil under different management and tillage practices [29
Soil microorganisms effect the formation and stabilisation of soil aggregates, and thereby significantly involve themselves in the processes of building soil structure [31
]. Microbes release excretions, including extracellular polymeric substances, which enmesh soil particles into aggregates. Similarly, soil particles can also be enmeshed into aggregates by fungal hyphae [34
]. Fungi have been found involved in the binding of larger particles, and are predominantly responsible for stabilization of macro-aggregates due to their hyphae structure [27
]. The influence of fungi and bacteria on aggregate stabilization varies widely among species and depends considerably on the nature of the available substrates [37
]. In general, fungi are better correlated with aggregate stability and lead to stronger binding forces between soil particles than with bacteria [38
Soil physical structures and microbial community composition shift in short timescales (weeks) depending on environmental conditions, such as (soil-)climate and related soil ecosystem conditions, e.g., soil moisture. The extents of the shifts in soil abiotic and biotic properties differ depending on the crop and the management system of the cultivation [39
Overall, this indicates a seasonal connection between the soil fungal population and the soil structure, particularly the size and mass distribution of aggregates. Our aim was to explore this possible correlation between the soil fungal population and the soil structure in a case study for apple replant disease. This was done by analysing the sizes and mass distributions of soil sieve-size fractions, and their physical stability, and contrasting the results with the replant effects on tree vigour. For this, we analysed and compared the soils of an apple orchard where apples were cultivated on initially planted and repeatedly planted soils in the direct vicinity and under identical cultivation management. The data were collected over four time intervals in one growing season from March to October in 2018.
In this study we investigated correlations between the dynamics of soil fungal populations and soil structure (aggregates) in relation to a gradual impact of replanting on tree vigour in a series of time intervals over one growing season; we used not-replanted and replanted soil. Our results show reduced aggregate size and stability, along with decreasing density of total soil fungal DNA (ITS) and increasing density of Alternaria-group (Ag) for apple trees repeatedly planted on the same site, which suffered a loss of vigour. We found that the density of Ag and soil structure parameters correlate at replant-indicative time intervals—in our study observed in August.
The determination of total fungal densities highlights a replant-related effect in June and August (and shows no replant-related specific behaviour of the total fungal population in March, April or October). One replant-responsive soil fungal group, exemplary for indicating shifts in the soil fungal community, the Alternaria
-group (Ag) (class Dothideomycetes
, order Pleosporales
, family Pleosporaceae
], continuously increased its density in replant soil over the growing season, resulting in a distinct difference of Ag density between soils in August. On the same site, the Ag was found to be replant-indicative by density of soil fungal population two years earlier (2019) [19
]. However, in 2016 an increased Ag density under replant conditions was observed in April with a two-fold greater Ag density in no-replant soil and a four-fold greater Ag density in replant soil as compared to Ag densities found in April 2018. Inter-annual variations have also been reported for the date of maximum growth difference between treated and non-treated replant soils, and for the effect of soil treatments regarding combating replant-affecting soil microbes [61
The formation of aggregates exhibits different dynamics between replant soil and no-replant soil during the growing season. While the degree of aggregation follows similar patterns, the aggregate formation process is changed under replant conditions. The steady degree of aggregation between April and August suggests that aggregate turnover processes are prevented over summer under replant conditions. A decreased aggregation of replant soil has previously been reported for the replanting of peaches (Prunus persica
]. Concomitant with our results, the authors showed that the replant-specific low aggregation was due to a decreased proportion of fraction 2000–6300 µm and increased proportions of fractions 125–250, 250–500 and 500–1000 µm under replant conditions.
Our results show that fractions from 125 to 1000 µm and fraction 2000–6300 µm are replant-sensitive. In contrast, fractions ≤ 125 µm and 1000–2000 µm are replant-inert. The measurements of the mass distribution of sieve-size fractions highlighted differences in the composition of soils regarding soil structures (aggregates) in the replanted and initial planting area, which are significantly pronounced in August and less strong in October. Our indications of replant-sensitive sieve fractions in size ranges of fine sand (125–250 µm), medium sand (250–500 µm) and coarse sand (500–1000 µm), though not fine sand to silt and clay (≤125 µm), are consistent with several studies that state greater replant-related tree vigour suppression in light sandy soils as compared to heavy clay or loamy soils [11
]. This result implies that aggregated particles in size range of small and large macroaggregates (sands) may perform as alternative microhabitats for increased densities of soil fungi.
According to our results, the mass distributions of fractions from 125 to 1000 µm, and fractions 2000–6300 µm are linked to increases of soil fungal densities (Ag) in August. The decrease of soil in fractions 2000–6300 µm and the increase of soil in fractions from 125 to 1000 µm, correlate with an increase in the density of soil fungi (Ag) under replant conditions. This observation is supported by a distinct, though not significant replant-specific behaviour of soil fungi also observed in June, but this could not be related to any change in the mass distributions of sieve-size fractions at that time of the year. Nevertheless, the observations suggest an interaction between soil fungi (Ag) and the formation of soil structures (aggregates) during summer. Soil fungi, in particular filamentous fungi, have a well-documented impact on soil structure by formation or disintregration of aggregates, especially of macroaggregates (>250 µm) [64
]. The relevance of the correlation analysis is the consideration of potential interactions between soil and the apple under replant conditions over time. The ecology of the soil fungal populations and their association with the soil structure may be the next step in understanding causal interlinkages related to replant disease. For this purpose, we understand our case study as a first step that needs to be further tested by annual duplication in repeated studies in the field.
Wet-sieving and ultrasonication highlighted an increased concentration of less stable soil structures in fractions from 125 to 1000 µm in replant soils in August. Less stability of fractions 250–5000, 500–1000 and 2000–4000 µm, though not in fraction 1000–2000 µm, has previously been reported for the replanting of peaches (Prunus persica
]. Other observations of replant-specific proportions of aggregates with differing stability, notwithstanding 2 h sterilisation due to autoclaving [30
], showed a high persistence of aggregate structures under replant conditions, even under extreme abiotic conditions. Persistence of water-stable aggregates for decades to centuries was already proven by Jastrow (1996) [66
]. The potential persistence of a replant-sensitive aggregate stability class of WS ≤ 50 J mL−1
could contribute to the strong persistence of replant-effects that have been observed also after grubbing and irrespective of catch crops [67
Aggregate-disintegrating processes in smaller and less stable aggregates have been found at a high Ag density. Increased Ag density is linked to less stable aggregates and tree vigour suppression in replant soil. The correlation between Ag density, soil structure and tree vigour means that replant-effects can pass unnoticed for most of the vegetation period and become obvious only in specific time intervals. This is relevant for monitoring replant effects by parameters of the soil.
A steady increase of replant-effects on soil parameters in the summer season may potentially match with the sensitive stage of apple nutrition by root performance. For apple understock M.9, steadily increased growth of root has been reported from June until August [68
]. Root flush has also been reported around bloom [68
], approximately in late April to May in Germany [70
], in line with replant-specific increased Ag density observed in April 2016 [19
]. Interestingly, the soil parameters’ return to a similar density as was determined at the beginning of the growing season in March before the beginning of dormancy season in October, and then did not differ between no-replant soil and replant soil anymore. This suggests that a replant-effect based on a shifted quantitative composition of soil fungal population is in competition with root growth in soil and de facto diminishes tree vigour by an offset of ontogenetic development, probably due to seasonally limited access to nutrients.
Our observations suggest that differences between replant and no-replant soils are pronounced, but may occur at irregular intervals. This in turn would mean that continuous and densely gridded monitoring of soil (and plant) during the whole growing season of apple would be necessary to detect indicative parameters for apple replant disease. It also shows that one-time sampling of orchard test sites and homogenised soil samples taken at few times only can be misleading in detecting interactions of soil fungi and soil structure (and tree vigour), depending on the time of sampling.
An analysis of interrelations between soil fungi, soil structure and apple tree vigour (suppression) will require continuous and densely gridded monitoring of soil to detect replant effects at indicative time intervals, and needs to be performed on single planting spots rather than with homogenised soil samples.
Soil structure was found to be replant-sensitive by mass distribution of large and small macroaggregates (2000–6300 µm, from 250 to 1000 µm) and large microaggregates (125–250 µm). Small macroaggregates and large microaggregates are less stable under replant conditions. The statistical analyses suggest that specific replant-responsive soil fungi, here the Alternaria-group (Ag), are involved in replant-related changes in soil structure. Hence, replant-specific aggregate-disintegrating processes seem to be related to densities of soil fungi. A correlation between soil fungi and structure can only be detected at specific time intervals over the growing season. Pronounced differences in soil structure between no-replant soil and replant soil occur together with a selective growth of Ag densities in late summer.
The density of replant-responsive soil fungi (Ag), in particular, is highly correlated with the plant reaction of trees in replant soil, so we conclude that the replant effect is a biologically active process. On the one hand, changes in soil structure contribute to the functional conditions for growth of specific soil fungi, and on the other hand, soil fungi may be involved in the formation of less stable soil aggregates. Our study suggests that the interaction between soil fungi and soil aggregates may be causally linked to interrelations between replant soil and plants.
An analysis of the interrelations between soil fungi, soil structure and apple tree vigour (suppression) will require continuous and densely gridded monitoring of soil to detect replant effects at indicative time intervals, and needs to be performed on single planting spots rather than with homogenised soil samples. In an applied context of the restoration of replant soil, our results provide the first indication that a potentially negative effect of the Ag on soil structure could be managed by good soil aggregators, e.g., mycorrhiza, to restore soil structure under replant conditions.