Preliminary Findings of New Citrus Rootstocks Potentially Tolerant to Foot Rot Caused by Phytophthora

: Phytophthora spp. are one the most common soil-borne pathogens in citrus crops, in which Phytophthora nicotianae and P. citrophthora are the most relevant species, causing disease problems worldwide, such as foot rot and gummosis of the trunk, branch canker, brown rot of fruit, feeder root rot in orchards, and seedling damping-off in nurseries. Phytophthora -tolerant citrus rootstocks are essential for its control and for the success of the citrus industry. The aim of this study was to determine the susceptibility of new citrus rootstocks with low HLB incidence to Phytophthora diseases. Thus, plants of several commercial and new citrus rootstocks originating in different breeding programs were inoculated with an isolate of P. nicotianae . Thirty days post inoculation (DPI), the damage of lesion length in stem was measured for each plant. These results displayed a different susceptibility response to the damage caused by P. nicotianae among the citrus rootstocks tested. Thus, eleven new citrus rootstocks (B11R3T25, B11R5T25, B11R5T49, B11R5T60, B11R5T64, N40R1T18, N40R1T19, N40R3T25, WGFT + 50-7, UFR-6, and CL-5146), which have not been previously studied against Phytophthora diseases, improved the tolerance effect of Carrizo citrange. Our ﬁndings provide useful information for citrus growers on rootstock selection to address incidence problems caused by Phytophthora spp.


Introduction
The citrus industry has a great influence on Spanish agriculture. Spain is the top citrus producing country in the European Union and the sixth in the world, with a total production of over six million tons [1].
Phytophthora spp. are the most serious soil-borne pathogens of citrus trees worldwide, and they usually inhabit these crop soils. Ten species of this genus have been described as the causal agents of several citrus diseases, such as foot rot and gummosis of the trunk, branch canker, brown rot of fruit, feeder root rot in orchards, and seedling damping-off in nurseries [2][3][4][5][6][7]. Nevertheless, P. nicotianae (syn. P. parasitica) Dastur and P. citrophthora (R.E. Sm. & E.H. Sm.) Leonian are the most widespread and relevant species worldwide [5].
Foot rot and gummosis disease infection processes increase under rainy conditions with warm temperatures (20-25 • C). Phytophthora propagules can splash from the soil to the trunk through rain drops near the ground level, infecting wounds or growth cracks that produce plant tissue lesions along the trunk [8]. These infections typically initiate at the base of the trunk. Typical symptoms of this disease in trees include growth flush reduction, defoliation, and twig dieback. When the disease is aggressive, infected trees can die, which occurs more frequently in younger than old trees [6].
Phytophthora spp. management has usually been carried out with systemic fungicides from the group of phosphonates (Fosetyl-Al) and phenylamide (metalaxyl), which generate colonization protection against P. nicotianae and P. citrophthora for three months [9,10]. However, new environmental trends in European agriculture are conducive to reduce the use of synthetic pesticides and lowering the presence of residues in food, with greater safety from chemical products for growers, and to avoid the emerging risk of pathogen resistance [11,12], such as species of Phytophthora [13]. In this sense, there is an additional need to shift agriculture towards sustainable methods that reduce supplies and costs.
In addition, cultural practices can be implemented in citrus orchards to reduce the incidence of diseases caused by Phytophthora spp. Thus, citrus growers apply different techniques to reduce excess water in the soil by means of drainage practices or an optimal irrigation dosage, as rainy and waterlogging conditions foster growth of this pathogen and dispersion and infection of its propagules to plant material [14]. These techniques play an important role in reducing propagation and development of the pathogen zoospores (main infective propagules) and mycelia, respectively. While Phytophthora spp. can produce other types of propagules, such as chlamydospores and oospores, which are potentially infective for plants, they mainly operate as latent structures of survival with a viability lasting several years. Thus, the use of pathogen-free plant material and disinfection of agricultural equipment and soil are required to avoid the occurrence of these resistant propagules [15]. However, all these cultural techniques are not fully implemented by growers and entail time-consuming and laborious works.
On the other hand, a proper choice of healthy and Phytophthora spp.-tolerant citrus rootstock has been reported as an essential factor to avoid these diseases in newly established orchards, as the rootstock confers tolerance to the whole plant against this pathogen [16]. Trifoliate orange (Poncirus trifoliata (L.) Raf.) and its hybrid Swingle citrumelo (Citrus paradisi x P. trifoliata) have been described as tolerant to P. nicotianae; nevertheless, they are not demonstrated to be tolerant against P. palmivora. On the other hand, Citrus volkameriana is also reported as tolerant to P. nicotianae [17], and Carrizo citrange (Poncirus trifoliata x Citrus sinensis), which is the most commonly cultivated rootstock in Spain (approximately 61% of citrus orchards) [18], is described as having an intermediate level of tolerance to Phytophthora spp. [19]. On the contrary, other citrus rootstocks, such as Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and Rough Lemon (Citrus jambhiri), are described as Phytophthora spp.-sensitive rootstocks [19,20]. In addition, the occurrence risk of emerging diseases, such as Huanglongbing or citrus greening disease (HLB), have increased in the Iberian Peninsula, due to the dispersal in Spain and Portugal of Trioza erytreae [21,22], which is one of the most important insect vectors of the HLB causal agents [23]. Currently, a wide range of commercial citrus rootstocks are available to citrus growers, and in recent years breeding programs are generating new plant material for the citrus industry. To our knowledge, many of these new citrus rootstocks with reported low disease incidence of HBL have not been evaluated against Phytophthora diseases yet. The main aim of this work was, therefore, to assess tolerance to foot rot caused by Phytophthora in new citrus rootstocks originating in different breeding programs.

Plant Material and Experimental Design
Fourteen-month-old citrus plants belonging to twenty-four different rootstocks from in vitro culture were provided by the Agromillora Group nursery (Subirats, Barcelona, Spain) ( Table 1). The study was divided into two experiments due to the high plant number and production. Each experiment was carried out in the 2020 and 2021 spring seasons, respectively, as previous described [24], under greenhouse conditions (26 • C average temperature and 96% average relative humidity) located at the Las Torres Center in the Andalusian Institute for Agricultural and Fisheries Research and Training (IFAPA) in Alcalá del Río, Seville, Spain (37 • 30 43.3" N; 5 • 57 47.4" W). For each experiment, plants from each rootstock were separated into two treatments [inoculated and control (non-inoculated) plants], with eight replicates for inoculated plants and eight plants for non-inoculated. In all experiments, Carrizo citrange was used as the reference-comparative rootstock. Each plant was grown in 1.6-L pots with a mix of one part of silica sand and two parts of peat moss substrate (Sphagnum moss, wood fiber, and perlite; Gramoflor; Vechta, Germany) kept under an acclimation period of eight months and irrigated with water thrice per week depending on water requirements, non-nutritive solution was applied. Before starting the experiment, plants were distributed under a randomized block design.

Isolate of Phytophthora
The isolate of Phytophthora (Pn1) was supplied from the fungal collection of Instituto Valenciano de Investigaciones Agrarias (IVIA). DNA identification was carried out to confirm the genus and specie of this oomycete. First, the isolate was grown over cellophane membrane on Petri dishes (60 mm of diameter) with potato-dextrose-agar (15 mL, PDA, Biokar diagnostics, Solabia Group, Cedex, France) and incubated under chamber conditions (25 • C in darkness) for ten days. Next, the isolate was sent to a commercial laboratory (Agricultura y Ensayo S.L.; Alcalá de Guadaira, Seville, Spain) for DNA extraction, PCR amplification, and sequencing. Briefly, DNA extraction process was carried out following the manufacturers' instructions of HigherPurity™ Plant DNA Purification Kit (Canvax Biotech, S.L., Cordoba, Spain). PCR amplification and further sequencing of the Internal Transcribe Spacer (ITS) region of the nuclear rDNA was carried out with ITS1 and ITS4 primers [36]. The PCR reactions were mixed in a total volume of 25 µL containing DNA product, 0.8 mM dNTPs, 2.5 mM MgCl2, 1X PCR Buffer, 0.75 µM of each primer, and 0.05 U/µL of Horse-Power-Taq DNA polymerase (Canvax Biotech, S.L., Cordoba, Spain), conducted in a BT1 Thermocycler (Whatman Biometra, Göttingen, Germany) with an initial step of denaturation at 95 • C for 5 min, followed by amplification of 35 cycles of 30 s at 95 • C, annealing 45 s at 56 • C, and extension at 72 • C for 2 min. The amplified fragments were visualized by electrophoresis in 2% agarose gel stained with RedSafe (iNtRON, Biotechnology, Inc., Korea). The PCR products were sequenced by a DNA commercial sequencing service (Secugen, S.L., Madrid, Spain). The raw sequences were edited using the Chromas 2.6.4 program (Technelysium Pty Ltd., South Brisbane, Australia), assembled by the DNAMAN 6.0.3.93 program (Lynnon Corporation, San Ramon, CA, USA) and compared with sequences from GenBank genetic sequence database using BLAST (version 1.17, Basic Local Alignment Search Tool, National Center for Biotechnology Information).

Inoculation Process
For the inoculation process in all experiments, Pn1 was grown for refreshing on 15 mL PDA Petri dishes and later incubated under chamber conditions for two weeks. Then, the groups of fourteen-month-old citrus plants were inoculated with Pn1. Disks of bark 5 mm in diameter were incised and cut from the stem of each plant with a sterile cork borer (5-mm diameter) at 30-35 cm above the ground from the rootstock trunks. Mycelial disks (with the same size and tool) were cut from the active PDA culture of Pn1 ( Figure 1A) and inserted mycelial face inward into each hole stem section for inoculated plants. PDA disks (with the same size and tool) from non-cultured PDA petri dishes were cut and placed in each sliced stem section for control plants (non-inoculated). Each agar disk was covered with the bark disk ( Figure 1B), held, and wrapped in place manually with a strip of cotton moistened with sterile water, and then covered around each stem with aluminum foil ( Figure 1C). All inoculated and control plants were incubated under greenhouse conditions, and cotton layers were periodically watered for three days a week to keep the inoculum moist.
step of denaturation at 95 °C for 5 min, followed by amplification of 35 cycles of 30 s at 95 °C, annealing 45 s at 56 °C, and extension at 72 °C for 2 min. The amplified fragments were visualized by electrophoresis in 2% agarose gel stained with RedSafe (iNtRON, Biotechnology, Inc., Korea). The PCR products were sequenced by a DNA commercial sequencing service (Secugen, S.L., Madrid, Spain). The raw sequences were edited using the Chromas 2.6.4 program (Technelysium Pty Ltd., South Brisbane, Australia), assembled by the DNAMAN 6.0.3.93 program (Lynnon Corporation, San Ramon, CA, USA) and compared with sequences from GenBank genetic sequence database using BLAST (version 1.17, Basic Local Alignment Search Tool, National Center for Biotechnology Information).

Inoculation Process
For the inoculation process in all experiments, Pn1 was grown for refreshing on 15 mL PDA Petri dishes and later incubated under chamber conditions for two weeks. Then, the groups of fourteen-month-old citrus plants were inoculated with Pn1. Disks of bark 5 mm in diameter were incised and cut from the stem of each plant with a sterile cork borer (5-mm diameter) at 30-35 cm above the ground from the rootstock trunks. Mycelial disks (with the same size and tool) were cut from the active PDA culture of Pn1 ( Figure 1A) and inserted mycelial face inward into each hole stem section for inoculated plants. PDA disks (with the same size and tool) from non-cultured PDA petri dishes were cut and placed in each sliced stem section for control plants (non-inoculated). Each agar disk was covered with the bark disk ( Figure 1B), held, and wrapped in place manually with a strip of cotton moistened with sterile water, and then covered around each stem with aluminum foil ( Figure 1C). All inoculated and control plants were incubated under greenhouse conditions, and cotton layers were periodically watered for three days a week to keep the inoculum moist.

Disease Evaluation and Data Analysis
In all experiments, the aboveground symptoms of foot rot disease caused by Pn1 were evaluated for each citrus plant and treatment 30 DPI from the beginning of the experiment (inoculation day) ( Figure 2). Next, all the wrappings were removed, all the rootstocks stems were cut 25-30 cm above and below the inoculation site, and each lesion length caused by Phytophthora inoculum was measured using an electronic digital slide gauge (Absolute digimatic caliper, Mitutoyo Corporation, Kawasaki, Japan). The level of rootstock tolerance was evaluated by comparing the lesion length of each. Thus, the values of lesions lengths were statistically analyzed using the free software R version 4.0.2 [37], performing Horticulturae 2021, 7, 389 5 of 10 one-way ANOVA and LSD-Fisher tests (p < 0.05) for each experiment [38] with the package "agricolae" [39].

Disease Evaluation and Data Analysis
In all experiments, the aboveground symptoms of foot rot disease caused by Pn1 were evaluated for each citrus plant and treatment 30 DPI from the beginning of the experiment (inoculation day) ( Figure 2). Next, all the wrappings were removed, all the rootstocks stems were cut 25-30 cm above and below the inoculation site, and each lesion length caused by Phytophthora inoculum was measured using an electronic digital slide gauge (Absolute digimatic caliper, Mitutoyo Corporation, Kawasaki, Japan). The level of rootstock tolerance was evaluated by comparing the lesion length of each. Thus, the values of lesions lengths were statistically analyzed using the free software R version 4.0.2 [37], performing one-way ANOVA and LSD-Fisher tests (p < 0.05) for each experiment [38] with the package "agricolae" [39].

Molecular Identification of Phytophthora Isolate
The oomycete isolate (Pn1) used in this study was corroborated as P. nicotianae by sequencing of the ITS region. These sequences showed 96.78% identity with P. nicotianae upon BLAST match analysis (Supplementary Material).

Molecular Identification of Phytophthora Isolate
The oomycete isolate (Pn1) used in this study was corroborated as P. nicotianae by sequencing of the ITS region. These sequences showed 96.78% identity with P. nicotianae upon BLAST match analysis (Supplementary Material).

Discussion
Our findings provide information about the tolerance response to foot rot lesion length in the stem caused by P. nicotianae among new different citrus rootstock, which is described as one of the most relevant Phytophthora species causing citrus diseases, such as foot rot and gummosis of the trunk [6,40]. To our knowledge, the disease incidence caused by Phytophthora on new citrus rootstocks has not been researched recently.
Prior studies have reported Carrizo citrange as having known tolerance and weakness against P. nicotianae and P. palmivora, respectively [16]. Otherwise, other authors have described an intermediate tolerance to diseases caused by Phytophthora spp. However, five recently obtained citrus rootstocks (N40R2T19, WGFT + 50-7, B11R3T24, B11R5T25, and N40R3T25) improved the tolerance behavior of C. macrophylla in the 2020 experiment (Figure 3). In the latest experiment, Forner-Alcaide No. 5 rootstock improved the tolerance response of Carrizo citrange, but five others recently obtained citrus rootstocks

Discussion
Our findings provide information about the tolerance response to foot rot lesion length in the stem caused by P. nicotianae among new different citrus rootstock, which is described as one of the most relevant Phytophthora species causing citrus diseases, such as foot rot and gummosis of the trunk [6,40]. To our knowledge, the disease incidence caused by Phytophthora on new citrus rootstocks has not been researched recently.

Conclusions
Cultivation of citrus rootstocks tolerant to diseases caused by Phytophthora spp. is the most effective methodology to reduce these damages. Our results provide preliminary helpful information for citrus growers to perform an accurate selection of rootstocks in those areas where Phytophthora causes serious damage to citrus crops. Thus, this first screening work indicates that eleven new citrus rootstocks, namely, B11R3T24, B11R5T25, B11R3T53, B11R5T60, B11R5T64, N40R1T18, N40R1T19, N40R3T25, WGFT + 50-7, UFR-6, and CL-5146, are potential candidates to be taken into account to solve problems triggered by these diseases. These preliminary results are helpful for the citrus industry to increase the rootstock variability of their orchards, and for the research community and breeding programs aiming for future improvements in this field. Consequently, further studies will involve the field susceptibility of all twenty-four citrus rootstocks against damages caused by Phytophthora spp.