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Alleviation of Nematode-Mediated Apple Replant Disease by Pre-Cultivation of Tagetes

: Apple replant disease (ARD) is a severe problem in orchards and tree nurseries caused by yet unknown soil biota that accumulate over replanting cycles. This study tested the contribution of nematodes to ARD, and cultivation of Tagetes as a control option. In a pot experiment, Tagetes patula or Tagetes tenuifolia were grown in ARD soil, incorporated or removed. Nematodes extracted from untreated ARD soil and washed on 20 µm-sieves induced ARD symptoms when inoculated to apple saplings growing in a sterile substrate. In contrast, nematodes from Tagetes treated ARD soil did not reduce root growth compared to uninoculated plants, irrespective of Tagetes species and incorporation. In plots of five apple tree nurseries or orchards, either Tagetes or grass was grown on ARD soil. Nematodes extracted from the grass plots and inoculated to apple saplings significantly reduced plant growth compared to nematodes from Tagetes plots for all five farms. Apple rootstocks showed overall a significantly higher increase in shoot base diameter when grown on Tagetes -treated plots compared to grass plots, while this effect differed among farms. Plant-parasitic nematodes were too low in abundance to explain plant damage. In conclusion, the free-living nematodes involved in ARD can be controlled by Tagetes .


Introduction
Apple replant disease (ARD) has been recognized throughout pome fruit production regions of the world and has been studied extensively [1][2][3][4]. When establishing a new orchard on replant sites, trees commonly exhibit poor growth that shows in the significantly reduced shoot growth, necrosis and patchy blackening of root cells, impaired root hair development, and low cell vitality, which may lead to root death [5]. As a result, fruit yield and quality are significantly reduced [6]. Although consensus regarding the causality of replant disease has not been fully realized, mitigating measures such as soil pasteurization or fumigation significantly improved the growth of apple plants which gives evidence that the disease is caused by biotic factors [7]. Originally, replant disease, also known as soil fatigue, was described as the phenomenon that soil gradually looses its evaluated in field trials at a diverse selection of tree nurseries and apple growing farms, and the effect on plant-parasitic and non-parasitic nematodes was investigated. In biotests, the effect of nematodes extracted from Tagetes treated and untreated ARD soils on growth and ARD symptoms of susceptible apple plantlets was investigated. Furthermore, we tested the efficiency of different Tagetes species and soil incorporation on ARD in pot tests.

Pot experiment on the effect of Tagetes with or without incorporation into soil on nematode-mediated ARD
In a pot experiment, we investigated whether nematode communities recovered from ARD soil affect apple plant growth and whether preculture of Tagetes in ARD soil reduces the negative effect of the nematode community on apple plant growth. Since different Tagetes species produce different biocidal compounds, which may be exuded or not from the root, we tested whether T. patula and T. tenuifolia differ in their effect on nematode-mediated ARD, and whether incorporation of Tagetes into the soil is necessary for mitigation of nematode-mediated ARD. The soil was obtained from a field in the Pinneberg area near Heidgraben, Germany (53°41´57.1´´N 9°40´59.4´´E). Since 2009, the rootstock of the cultivar 'Bittenfelder Sämling' was planted repeatedly in two-years cycles [46]. The soil was sampled around the roots of plants at a depth of 0-30 cm and sieved through a 5mm mesh. Samples were stored at 4°C for 1 week before the pot experiments. Seeds of T. patula 'Single Gold' or T. tenuifolia (Saatzucht Bardowick, Bardowick, Germany) were nursed separately in sterile sand. Six two-week seedlings were transferred into each pot filled with 1 L of the ARD soil. Plants were grown for 8 weeks until flowering. To investigate the effect of plant incorporation into soil, Tagetes plants were removed from 20 pots, chopped into 0.5-cm pieces, and 50 g per pot was mixed with the soil. Tagetes in the rest of the 20 pots were removed and not incorporated into the soil. Control pots were not treated with Tagetes. After four weeks, nematode communities (plant-parasitic and freeliving) were extracted from each pot by centrifugal floatation using MgSO4 at 1.18 specific density [47]. Nematodes were collected on 20-μm sieves and washed with sterile water. Nematodes were inoculated to in vitro propagated and acclimatized, 5-weeks old M26 apple plantlets growing in 500 mL sterile sand, by equally distributing the suspension into four 5-cm deep 1-cm wide holes in 2 cm distance around the shoot. Pots were placed in a randomized complete block design in the greenhouse. The fertilizers Hakaphos NPK (+Mg) [15:10:15(+2)] (Compo, Münster, Germany) (0.5 g per pot) and 36% calciumcarbonate (Vereinigte Kreidewerke, Söhlde, Germany) (2 g per pot) were applied weekly. Plants were watered every 2-3 days as required. The greenhouse conditions were 22 ± 2.5°C, 60 ± 8.7% relative humidity, and a 16 h photoperiod. Pots were sampled eight weeks after inoculation to determine shoot length, shoot fresh mass, leaf fresh mass, and root fresh mass. Overall, the plant growth assay comprised 20 replicates of each treatment with nematodes from ARD soil treated with T. patula, T. tenuifolia, T. patula with incorporation, T. tenuifolia with incorporation, or from untreated ARD soil. Additional controls were plants grown in uninoculated sterile substrate, and plants grown in ARD soil.

Effect of Tagetes in mitigating ARD in apple growing farms and tree nurseries
Two apple-growing farms with either organic (farm M) or conventional (farm J) practice, and three tree nurseries either in Northrhein-Westfalia (farm L) or Schleswig-Holstein (farms S, C) were selected where apple plants were repeatedly replanted and problems with ARD were reported ( Table 1). The soils differed in texture, pH as well as N and C contents ( Table 2). In in-field trials, plots were either kept under grass to maintain the status of ARD, while avoiding soil erosion and weed growth, or cultured with T. patula 'Nemamix' (farms S, C, M, J) or T. erecta (farm L) for the vegetation period of 2019. Plants were chopped and incorporated by mulching into the soil before planting of apple ( Table  1). The effect of the Tagetes pre-treatment on apple plant growth was determined by measuring the increase in shoot diameter during the vegetation period of 2020. In the tree nurseries, the diameter of 40 rootstock plants of the middle rows of each plot was taken at the base (above the soil) shortly after planting and in November before uprooting. In the apple orchards, trees (excluding border plants) were marked at 20 cm above the grafting, and diameter was measured twice (in orthogonal direction) each in May and November 2020.
To determine the contribution of nematodes to ARD, soil samples from all plots were collected in November 2020 at a depth of 0-20 cm with 6-8 sampling points between apple plants. The soil samples were stored at 4°C before nematode communities were extracted.
To determine the number of nematodes of plant-parasitic genera and non-parasitic nematodes, 250 mL soil aliquots were extracted using an Oostenbrink elutriator [47]. Nematodes were collected on three mounted 45 µm sieves, washed into a beaker, and transferred onto an Oostenbrink dish to get a clean sample. After 48 h, the nematodes in the Oostenbrink dish were collected on a 20-µm sieve, transferred to 30 mL tap water, and counted on a counting slide under an Olympus SZX12 stereomicroscope at 40×-80× magnification (Olympus, Hamburg, Germany).

Biotest on ARD-induction by nematodes from apple plots with preceding Tagetes or grass cultivation
For the biotest, nematode communities were extracted from 1 L soil samples from apple plots with preceding Tagetes cultivation or control plots with preceding grass cover. The extraction was done by centrifugal floatation in MgSO4 at 1.18 specific density [47]. Nematodes were collected on 20-μm sieves and washed with sterile water. The nematodes were inoculated to apple plants grown in sterile sand and incubated in the greenhouse as described above. Shoot fresh mass, leaf fresh mass, root fresh mass, and shoot length were determined eight weeks after inoculation. Overall, the biotest comprised 10 replicates for both the Tagetes treated ARD soils and the grass treated ARD soils for each farm. Plants growing in uninoculated sterile sand served as control.

Statistical analysis
Statistical analyses were done using the GLIMMIX procedure of the software package SAS 9.4 (SAS Institute Inc., Cary, NC, United States of America). Plant growth parameters were analyzed with the assumption of a normal distribution without data transformation, which was checked by qq-and residual plots. For multiple comparisons, the Pvalue was adjusted by the method of Tukey (ADJUST=TUKEY in the LSMEANS statement). To account for overdispersion, degrees of freedom were approximated by the method of Kenward-Roger (DDFM=KENWARDROGER). To analyze the effect of Tagetes (TAGETES = 1 or 0) on shoot base increase (SBI) in plots of five farms, the generalized linear mixed model SBI = TAGETES FARM was used, with PLOT as a random effect. The effect for each farm was estimated by using contrasts (LSMESTIMATE statement). The effect of preceding Tagetes cultivation on the proportion of plant-parasitic nematodes (PPN) was tested in a generalized linear mixed model PPN / TOTAL = TAGETES with FARM as random effect, binomial distribution and Logit transformation, using GLIM-MIX. An effect was regarded as significant at the type III error P ≤ 0.05.

Effect of T. patula or T. tenuifolia, with or without incorporation into soil, on nematodemediated ARD (pot experiment)
Washed nematode fractions from differently treated ARD soil (Heidgraben field), with or without Tagetes preculture and with or without soil incorporation of Tagetes, were tested for their effect on the growth of roots and shoots of apple plants. The plants that received this nematode inoculum from untreated ARD soil, or that were directly planted in ARD soil discolorated roots. Overall, nematodes extracted from the different treatments significantly differed in their effect on the determined parameters of plant growth (MANOVA, P < 0.0001). Nematodes extracted from untreated ARD soil significantly reduced root fresh mass compared to nematodes from all Tagetes treatments of ARD soil (Table 3). Roots grown in ARD soil or grown in substrate with nematodes from ARD soil did not significantly differ in fresh mass, but were 44-68% smaller than in the control without nematodes. In contrast, apple roots inoculated with nematodes from the Tagetes treatments did not significantly differ from the control without nematodes. Incorporation of Tagetes was not a significant factor (P = 0.84), while root fresh mass had a trend to be increased in treatments with T. patula compared to T. tenuifolia (P = 0.0547), as revealed by the respective contrasts in ANOVA.
Shoot parameters were significantly reduced by nematodes from ARD soil compared to the control without nematodes (Table 3). Treatments with T. patula alleviated this effect, whether incorporated into soil or not. However, nematodes from ARD soil that was treated with T. tenuifolia did not significantly differ in their effect on shoot parameters compared to nematodes from untreated ARD soil. Incorporation of Tagetes was not a significant factor, as revealed by the respective contrasts in ANOVA (P > 0.08). Only T. tenuifolia, which produced more shoot biomass than T. patula, showed a trend for increased shoot growth in the biotest when treatments with and without incorporation are compared. Shoot parameters were most affected by direct growth in ARD soil compared to growth in substrate that was inoculated with nematodes.

Effect of preceding Tagetes cultivation on apple shoot growth in apple orchards and tree nurseries
In three apple orchards and two tree nurseries, the increase in apple trunk diameter over a season was compared among plots that have been precultivated either with Tagetes or with grass before planting apple rootstocks. In general, apple plants cultivated in Tagetes pre-cultured ARD soils grew significantly better than those in the untreated grass plots (ANOVA, P = 0.0004; Fig. 1). The trunk diameter at the base increased in grass plots by 2.3 mm and in Tagetes plots by 3.0 mm, on average. However, growth significantly differed among farms (P = 0.0001), and the effect of Tagetes was dependent on the farm (interaction effect FARM * TAGETES, P = 0.0001). The Tagetes effect was strongly evident in farms S and C, and less pronounced in farms J and L (Fig. 1). Also blocks of farm C where hollows cause temporary water logging showed a pronounced positive Tagetes effect on apple trunk growth. In farm M, conditions during this season did not allow for increase of the trunk diameter on average, thus no effect of Tagetes was realized.

Effect of preceding Tagetes cultivation on plant-parasitic genera and total nematodes in apple orchards and tree nurseries
The numbers of plant-parasitic nematodes in the apple orchards and tree nurseries were generally low, with 77 individuals per 100 ml soil on average ( Table 4). The genus Pratylenchus that was previously linked to ARD was only detected in low numbers in two tree nurseries, farms S and C. The genera Rotylenchus, Meloidogyne, or Trichodorus were each detected in two of the farms. Paratylenchus was the most abundant plant parasite and detected in three farms. None of the genera was detected in all ARD soils. In apple plots with preceding Tagetes cultivation, the proportion of plant-parasitic nematodes was sig-nificantly lower than in the ARD plots (P = 0.0002). Farm L was characterized by the highest number of non-parasitic nematodes, while only four Paratylenchus were found. A 16 % increment of the non-parasitic nematodes was achieved after pre-treatment with Tagetes erecta, while Paratylenchus became undetectable. In farm S, the plant-parasitic nematodes were reduced by 97% and non-parasitic nematodes were reduced by 11% after cultivation of T. patula. In farm M, the non-parasitic nematodes achieved a 55 % increment after Tagetes treatment and Meloidogyne was reduced by 64%. In farm J, the Tagetes treatment increased the abundance of non-parasitic nematodes by 43% and the genus Paratylenchus by 36%. In soil of farm C, the non-parasitic nematodes were increased by 331% after the pretreatment with Tagetes while Rotylenchus was reduced by 42%. Figure 2. Discoloration and stunting of apple roots in the biotest of nematode fractions extracted from soils of plots in tree nurseries (farms L, S, C) and apple orchards (farms M and J), that were precultured with either Tagetes or grass before planting of apple rootstocks.

Biotest with inoculation of nematodes from apple plots with preceding Tagetes or grass cultivation
Nematodes from plots that were not treated by Tagetes before replanting of apple rootstocks caused browning and size reduction of roots of inoculated apple plantlets, which is typical for ARD (Fig. 2). A two-factor ANOVA revealed significantly higher root fresh mass of plants that were inoculated with nematodes from Tagetes-treated ARD soils compared to grass-treated ARD soils (P = 0.0001). The factor FARM also had a significant effect on root fresh mass (P = 0.0001). The interaction of FARM and TREATMENT was not significant (P = 0.14). Contrasts revealed for each farm a significant Tagetes effect on root fresh mass in the biotest (P < 0.007), while the difference between treatments was most pronounced for farm L, followed by farms S and M (Fig. 3).  Shoot fresh mass and shoot length were significantly larger when plants were inoculated with nematodes from the Tagetes treated ARD plots compared to inoculation of nematodes from the grass treated ARD plots (P = 0.0001, Table 5). The factor FARM had a significant effect on shoot fresh mass (P = 0.0089), but not on shoot length (P = 0.33). The respective interactions of FARM and TREATMENT were not significant. Contrasts revealed for each farm a significant Tagetes effect on shoot fresh mass (P < 0.006), while the difference between treatments was most pronounced for farm L, followed by farm S (Table 5).
In this study, the efficacy of Tagetes preculture to improve apple plant growth in ARD soils has been shown in field trials of a diverse selection of orchards and tree nurseries. All studied farms had major problems with ARD, especially the tree nurseries that typically replant apple rootstocks every or every second year, depending on their specialization on rootstock production or grafting, respectively. The seasonal increase in shoot base diameter on Tagetes plots was on average 30% higher than on adjacent control plots where grass was grown instead (Fig. 1). This effect was pronounced for the tree nurseries, while the apple-growing farm J showed the same trend. The other apple-growing farm experienced unfavourable conditions during the season, so that the stems did not show detectable increase in diameter. Therefore, an effect of preceding Tagetes cultivation on stem growth could not be detected there. However, when nematodes were extracted from these plots and inoculated to susceptible M26 apple plantlets, the effect of the preceding Tagetes cultivation on how the nematodes affected apple roots became very clear for all farms (Fig. 3). In contrast to the nematode fraction from Tagetes plots, the nematodes from ARD plots with preceding grass cultivation caused the discolorated and stunted roots that are typical for ARD (Fig. 2). This confirms our previous finding that the nematode fraction contained one main driver of ARD [31]. As in this study, the nematode fraction was obtained from soil by floatation on a dense MgSO4 solution, and collection and washing on a 20 µm-sieve. The nematode fraction thus contains the microbes that are associated with the nematode bodies. These microbes were shown to synergistically enhance ARD symptoms together with the nematode fraction, but they had hardly any effect when inoculated to apple plantlets alone [31]. While floatation is a standard technique to retrieve nematodes from soil [48], and the fraction on the 20 µm-sieve mainly contains nematodes when microscopically analysed, it might contain other small organisms and microbes associated with small organic particles, which may play a role in ARD. This needs to be ruled out in future studies. Neither in the previous nor in the present study played plant-parasitic nematodes a significant role in ARD. They were only detected in low numbers in the ARD soils. None of the genera was detected in all farms and could be associated with ARD. The genus Pratylenchus was only detected in two tree nurseries. It has been frequently reported in association with ARD [14,29,[49][50][51], with a damage thresholds of 50 Pratylenchus per 100 ml soil [52]. However, in other studies the reduction of Pratylenchus by nematicides did not improve tree growth in ARD affected orchards [32], or was hardly correlated with the gain in plant growth (R 2 = 0.186) [53], or ARD symptoms were observed despite reduction of Pratylenchus by soil treatment [30,52]. In an orchard in Washington, a heavy ARD infestation was treated either by fumigation or Brassica seed meal [54]. While Pratylenchus reached high densities in apple roots in the second year after fumigation but not in the Brassica seed meal treatment, this difference was not reflected by trunk increase or apple yield. Plant-parasitic nematodes thus can eventually increase damage of ARDaffected roots but did not have a relevant contribution to ARD at least in our study. Nevertheless, the Tagetes treatment in our study further reduced the relative abundance of phytoparasites within the nematode community, which is an added benefit. The count data showed that Tagetes changed the structure of the nematode community, while the total number of nematodes in soil tended to increase (Table 4).
In the pot experiment, both T. patula and T. tenuifolia significantly reduced plant damage by the nematode fraction from the treated ARD soil that was inoculated to apple plantlets. For the T. patula treatment, nematode-mediated ARD was abolished, as shoot and root growth did not significantly differ from the uninoculated control. The T. tenuifolia treatments were not equally efficient. Shoot growth was less improved compared to the T. patula treatments, and roots also showed this trend. The farms applied mainly T. patula to reduce ARD, but farm L used T. erecta instead. Notably, farm L showed the most pronounced reduction of nematode-mediated ARD among the farms in the biotest (Fig. 3). In contrast, the effect of T. erecta preculture on stem growth in the field was not significant for this tree nursery, while the other two nurseries significantly improved stem growth after T. patula preculture. This coincided with the overall lower plant growth in farm L compared to the other two tree nurseries. It should considered that the rootstock A2 cultivated by farm L may be more susceptible to ARD than M9. In farm M, lack of trunk growth in the field completely prevented the detection of a Tagetes effect, while the biotest clearly revealed a significant treatment effect. The farms differed in the type of cultured rootstock, soil type, soil texture, and other parameters, so that the differential effect of the Tagetes species might be better reflected by the growth of apple plants in the more controlled biotest. T. patula was reported to be attractive for root invasion by P. penetrans and other endoparasites, but the nematodes cannot multiply in the roots [55]. Cultivation of Tagetes as pre-crop reduced populations of root-knot and lesion nematodes and substantially increased yield in the subsequent crop (melon, tomato, or potato) [56,57]. This led to the view that Tagetes acts as a trap crop. However, metabolites released from the roots of mature Tagetes plants act against diverse herbivorous and non-herbivorous nematodes, especially thiophene compounds like α-terthienyl [58][59][60]. A study on the amount of nematicidal thiophenes in roots of Tagetes species revealed the highest concentration in T. tenuifolia, followed by T. patula and T. erecta [59]. Interestingly, when comparing the amount of thiophenes produced by the roots per area of cultivated Tagetes, then probably T. minuta has the highest effect on nematodes, followed by T. patula, T. erecta, and T. tenuifolia (320 / 39 / 14 / 12 mg m -2 , respectively). This coincides with the slightly better performance of T. patula compared to T. tenuifolia in our pot experiment.
At the farms, Tagetes was incorporated into soil either in autumn or in spring. It is unclear whether soil incorporation of Tagetes contributes to the effect on ARD. In the pot experiment, incorporation of T. patula had no effect on growth of apple plantlets in the subsequent biotest with the inoculated nematode fraction. The treatment with T. tenuifolia showed a trend for increased shoot growth when the soil was incubated with the chopped and incorporated plants compared to the treatment without incorporation. This might be explained by a green manure effect on nematodes, rather than an introduction of nematicidal compounds from the shoot, because actively growing roots of Tagetes act most effective on nematodes while root extracts or other parts of the plant are less efficient [61,62].
Tagetes cultivation is already applied in tree nurseries to control P. penetrans. However, farmers are not yet aware that it is also a management option against ARD. Tagetes cultivation is less expensive compared to physical soil disinfection, and less damaging to the environment compared to chemical soil fumigation. However, it needs to be investigated for how long Tagetes cultivation suppresses the ARD causing biota, and to what extent production losses during Tagetes cultivation is outweighed by the gain in soil health.  Data Availability Statement: Any data from this article are available on reasonable request from the corresponding author.