1. Introduction
Kiwifruit is a recently domesticated plant belonging to the family
Actinidiaceae and the genus
Actinidia, which contains ±54 species [
1]. However, two taxa,
A. chinensis var.
deliciosa and
A. chinensis var.
chinensis, dominate the kiwifruit industry [
2]. Breeders have shown an increasing interest in kiwifruit, and long-term breeding programs have recently resulted in the development of several cultivars [
3]. Consumption of kiwifruit has been increasing steadily, with the fruit recognised as a superfood, owing to its high vitamin C content. Kiwifruit is a highly profitable crop with a long shelf life suited to global trade [
4]. In Italy, the area planted with kiwifruit is currently 25,000 ha [
5].
Recently, a new and complex disease called Kiwifruit Vine Decline Syndrome (KVDS), also known as “Moria del kiwi”, has appeared in different kiwifruit growing regions, being reported initially in Italy and then worldwide [
6,
7]. This has led to significant yield and economic losses, around EUR 300,000,000 in 2020 [
8]. KVDS was initially observed in poorly drained soils where waterlogging conditions occur most easily [
9]. The abiotic factor of waterlogging is thought to favour the development of this syndrome [
10], although KVDS can occur in many soil types. Several soil-borne pathogens have been implicated as aetiological agents (biotic factors) [
10].
Phytophthora spp.,
Pythium spp.,
Phytopythium spp.,
Fusarium spp.,
Cylindrocarpon spp. and
Desarmillaria tabescens were frequently isolated from affected plants [
9,
11,
12,
13]. Pathogenicity on kiwifruit cuttings has been demonstrated for some isolates:
Phytophthora cryptogea,
Phytophthora citrophthora,
Phytopythium vexans and
Phytopythium chamaehyphon [
7,
14]. Only a few bacterial strains, belonging to
Clostridium spp., have been identified as having a role in KVDS [
15].
The most dramatic symptom is a rapid, sudden, and irreversible wilting of the plant [
6]. The affected vines usually die within a few weeks, especially when evapotranspiration is the highest [
9]. Canopy symptoms are accompanied by severe damage to the root system, which generally displays a reddish discoloration, giving the roots a “rat-tail” appearance and almost complete decay of the white roots. KVDS can affect both young and mature plants of the species, and both
A. chinensis var.
deliciosa and
A. chinensis var.
chinensis under different orchard management practices, and commonly plants do not recover [
10].
Kiwifruit is usually not grafted in commercial orchards in Italy, but for its expansion into regions with heavy soils or other environmentally challenging characteristics, grafting of selected kiwifruit cultivars onto KVDS-resistant or -tolerant rootstocks might be essential for its future [
16], as already occurs for others woody crops grafted for the purposes of resistance or tolerance to different soil stresses (both biotic and abiotic) [
16,
17]. Previous work on kiwifruit has considered the compatibility of the most widely grown cultivar ‘Hayward’ with other species [
18] or has focused on micrografting methods and the evaluation of graft compatibility, shoot growth, and root formation and growth [
19]. The effect of rootstocks in promoting scion vigour has also been assessed [
20]. Other studies have evaluated rootstocks, focusing on abiotic stress, yield, vigour and yield components (production, quality, etc.) [
21,
22,
23]. There are only a few reports of resistance to specific soil-borne pathogens [
24], together with preliminary results concerning the resistance of
A. macrosperma to waterlogging conditions [
25].
In this study we investigated the main features of the root systems of six
Actinidia genotypes cultivated in four KVDS affected sites in North-East Italy. The test plants were monitored over two years particularly the radical system, as the root development can be affected by several factors, with genetics strongly influencing root growth and distribution, and chemical parameters, thus leading to resistance to soil-borne pathogens [
26]. For the aerial plant parts, only the total canopy biomass production was considered as these genotypes are intended for grafting or further breeding. The aims were to identify genotypes able to survive in KVDS-inducing soils and evaluate their agronomic performance in order to select the most promising rootstock candidates. This is the most desirable approach for establishing healthy and sustainable grafted kiwifruit plants.
4. Discussion
Some KVDS epidemiological traits are still under investigation, and at present there are no effective methods of control. Kiwifruit growers have empirically tried many strategies to maintain crop yields and their incomes [
43], but these strategies are not sustainable nor effective. The lack of effective control strategies results in continually increasing economic losses [
8]. The most promising approach, as with any soil-borne disease, is the use of tolerant or resistant species as rootstocks [
24,
44]. Within the Italian kiwifruit sector, this practice has not been widely used before. We have, therefore, screened genotypes of different
Actinidia species intended for use as rootstocks or for breeding programs for their putative resistance or tolerance to KVDS [
27,
29,
45,
46].
In the current research, six genotypes of
Actinidia spp. were planted at four infected experimental sites and tested for two years:
A. macrosperma (Ma176, Ma183 and Bounty71),
A. arguta Miss Green (MG), and
A. polygama (POL).
A. deliciosa Hayward (HW) was chosen as the susceptible genotype. At all of the experimental sites, KVDS dated back to 2014, and the sites were similar in their levels of KVDS infection and general features (
Table 1 and
Table 2). Consequently, the genotype behaviour recorded from each site was also similar. This allowed us to perform the experiment using four replicates per site, and this was confirmed by data normalization. In the statistical analysis, a one-way ANOVA was performed as a two-way ANOVA was not applicable because young plants grow quickly, so the year × treatment interaction would obviously be statistically significant (data not shown). Instead, changes between study years in the measured parameters were calculated as mean values because this provides a good indication of agronomic performance.
All the three genotypes of
A. macrosperma, Ma176, Ma183 and Bounty71, survived in KVDS-inducing soil, suggesting that they might be useful tools for providing resistance to the syndrome (
Table 3 and
Table 4). Furthermore, a genotype of known sensitivity, HW, proved to be susceptible and did not survive in the test soils (all plants were dead by the second year). For this reason, HW was not included in the second year of the study. Moreover, the POL genotypes died in the spring of the first year, indicating that this species is highly susceptible to KVDS. This result indirectly emphasizes the importance of biotic factors relative to environmental factors in KVDS aetiology [
6]. This is supported by the observation that the parents of the
A. polygama plants, as well as Hayward, used in the experiment are present in the germplasm collection of the University of Udine where KVDS is not reported (a few kilometers away from the experimental sites), and show excellent vegetative development and strong vigour, without any KVDS symptoms. The
A. arguta Miss Green plants showed no symptoms of KVDS in the first year, but in the second-year, root systems were affected (±25%), and consequently, many of the investigated root parameters were worse, with several plants dying. MG was affected at all experimental sites. This suggested that MG is not a resistant genotype but could be considered only tolerant. Consequently, where KVDS pressure was high, MG was not completely able to maintain a balanced root profile, nor was it fully capable of surviving.
A considerable range in kiwifruit root system agronomic parameters (biometric indices) was evident among the genotypes tested (
Table 5). For
A. polygama it was not possible to conduct any further analysis because all the plants died. Among the genotypes that survived the first year, HW had the worst performance. MG also performed poorly because by the second year it was affected by KVDS. In 2020, the best values were found in Ma176, and in the second year in Ma176 and Bounty71. Both Ma183 and Bounty71 showed strong increases in some indices (e.g., root volume) in the second year of the study, and Bounty71 in particular attained values that were similar to Ma176 during 2020. Bounty71 showed increases in all values. MG showed decreases in all the parameters due to KVDS. However, the small decreases in width and length of Ma176 and Ma183 were not significant.
The effect of soil-borne pathogens on protein degradation was clear (
Table 6) when considering the protein content in the resistant genotypes compared with the sensitive genotype (HW), as has been reported previously [
47]. Unsurprisingly, HW showed the greatest reduction in protein content, even when accounting for the data from the second year, with the remaining genotypes not noticeably affected. Once again, Ma176 tended towards the highest protein content; however, no statistical differences emerged among the tested genotypes. As previously discussed, there were no differences in protein patterns observed (
Figure S2).
Concerning the TNCs content (
Table 7), HW was the worst performing, further indicating its KVDS susceptibility. This parameter was clearly affected by KVDS and has known susceptibility to several biotic factors in many crops [
48]. Although the other genotypes tested showed higher values than HW, there were no statistical differences between any of the genotypes, or for the change in TNCs between years among the surviving genotypes. Nevertheless, we may assume from this assay that the genotypes that performed well will be able to accumulate more reserves in subsequent years, which is important for plant budburst, and flowering, etc. [
49,
50].
The mineral elements detected present in the root system (
Table 8 and
Table 9) showed some genotype specific effects. It is of great importance to understand their accumulation in the roots since they are further translocated to the aerial parts of the plant (i.e., leaves and fruits). At first glance, HW had significantly lower values for each element investigated than the other genotypes. However, no great differences emerged between the resistant genotypes. Nonetheless, in 2020, Bounty71 recorded the lowest Ca accumulation value. Calcium is involved in determining fruit size, firmness and sugar translocation [
51,
52], so we can speculate that this rootstock might eventually result in fruit size and quality reductions. During 2021, the trend continued, with Bounty71 accumulating less Ca and C than most of the varieties (the C levels were similar to MG). Indeed, the fact that Bounty71 recorded the lowest C concentrations among the surviving genotypes may mean that this cultivar is not capable of further accumulation of reserve substances or of maintaining a well-balanced whole plant profile in the long term [
53]. Regarding MG, despite it being affected by KVDS in the second year, it was surprising that the levels of mineral elements present in the root system tended to increase. We can assume that this was a concentration effect because the root volume of MG decreased in the second year of the study. Interestingly, the P content for MG in 2020 alone and for Ma183 in both 2020 and 2021 was significantly lower than that for the other genotypes. The P content should be carefully taken into consideration when selecting rootstock, because P is a component of essential molecules such as nucleic acids, phospholipids, and ATP (adenosine triphosphate), and is involved in controlling key enzyme reactions and the regulation of metabolic pathways [
54]. Finally, Ma 176 seemed to have the highest elemental concentration of all the genotypes, as did Bounty71 for the majority of elements, but these values were not statistically significant. Similar results were just obtained in preliminary experiments [
55,
56].
Considering canopy development measured in terms of dry weight (
Table 10), in both 2020 and 2021, Ma176 and Bounty71 had the highest values overall. In 2020, HW had a significantly smaller canopy than the other genotypes, and MG and Ma183 registered lower values than Ma176 and Bounty71 in both years. Perhaps owing to the effects of KVDS, canopy production in MG decreased in 2021. Finally, the changes in canopy dry weight between 2020 and 2021 were similar. We can report that the genotypes tested showed an interesting and different canopy development and appearance, which could be important for plant structure once grafting occurs, since the aptitude to develop canopy is an important factor for the whole aerial plant structure [
57]. In fact, a different canopy structure not only affects the aerial plant structure, but the biodiversity of the cultivated land as well (e.g., light competition is one of the most important factors that limits both the growth of plant species and the biodiversity of the soil under kiwifruit canopy) [
58].
As a final consideration, A. macrosperma and A. arguta showed better performance than HW and POL in every analysis carried out. Obviously, all the genotypes had different specific effects, but in the end, the majority demonstrated KVDS resistance.