Comparing Cotyledon, Leaf and Root Resistance To Downy Mildew in Radish (Raphanus Sativus L.)

Radish downy mildew (DM) is a disease caused by the oomycete Hyaloperonospora brassicae f. sp. raphani and it is a serious problem in radish production, an edible root vegetable crop of the Brassicaceae family. The objective of this research was to assess radish germplasm for DM resistance and to evaluate the response of different radish organs to the disease. Cotyledons, true-leaves and roots of 44 radish accessions were inoculated with H. brassicae isolates under controlled conditions. The cotyledons were individually evaluated 7dpi (days post-inoculation), and the leaves and roots 12dpi. DM symptoms varied with the radish genotype and plant organ analysed. Thirty-ve resistant and partially resistant accessions were identied and are promising sources to DM. A signicant correlation was observed between cotyledon and leaf (1 st and 2 nd leaves) DM resistance, but no correlation was found between the resistance of cotyledons or true-leaves and roots. Cotyledon and leaf response cannot be used to predict radish root resistance. However, cotyledon resistance has its own value because non-infected cotyledons will act as a barrier to slow disease progression to true-leaves and roots.


Introduction
Radish (Raphanus sativus L., n = 9) is a root vegetable of the Brassicaceae family, which includes the small or Western radish (Raphanus sativus var. sativus) and the daikon radish (Raphanus sativus var. longipinnatus), also known as Chinese oriental or Japanese radish traditionally used in East Asian cuisine. Daikon radish has similar growth requirements as Western radish, but the root has much larger and requires more space to growth. Depending on the cultivar, Longipinnatus radish group needs 50-80 days to harvest, requiring an early spring to mid-summer seeding date, because it is adversely affected by hot, dry weather and long days (APA 1988). On the other hand, Western radish varieties of Rhaphanus sativus var. sativus produce a much small root and reach harvest stage in 3-4 weeks.
Radish downy mildew (DM) is an economically important disease in main production areas worldwide (Glits 1977 Commercial varieties of radish are usually very susceptible to DM and chemical control does not provide an effective crop protection due to the short cultural cycle that would require fungicide spraying too close to harvest. The genus Hyaloperonospora (phylum Oomycota; family Peronosporaceae) is a group of biotrophic oomycetes responsible for DM disease in relevant crops from Brassicaceae family. DM in radish is caused by Hyaloperonospora brassicae f. sp. raphani, an airborne obligate pathogen strongly affected by temperature and air moisture. Favourable conditions for radish infection and disease dissemination are day and night moderate/cool temperatures of 20°C and 10-15°C, respectively, associated with high humidity (RH 80%) (Kofoet and Fink 2007).
The rst symptoms are yellow or brownish spots on the upper surfaces of radish cotyledons and mature leaves combined with a white sporulation on the corresponding abaxial epidermis. These spots eventually turn necrotic and the leaf dies. DM also infects radish roots that reveal a blackening area with H. brassicae sporulation, scarring and cracking, making them non-salable. Effective leaf disease control may prevent root damage because roots are infected by conidia that come from the leaves (Glits 1977).
The use of cotyledon evaluation to predict disease resistance in more advanced stages of plant development has the great advantage of being a faster and cheaper method requiring much less space, but for cotyledon evaluation to be effective, there must be a good correlation between the response of the cotyledons and the different organs with commercial value, which may not occur. However, cotyledon resistance in radish has commercial interest because young plants are harvested with the cotyledons, that must be exempt of disease, and may be important to decrease the progression of the disease to adult leaves and roots.
Integrated Pest Management (IPM) strategies combine different measures focused on a long term prevention of pest damages, including the adjustment of cultural practices, such as weed control (increase air circulation), keeping leaves dry avoiding overhead irrigation especially late in the day, removing plant debris after harvest, and also rotation with non-brassicas crops. H. brassicae pathogen persists as oospores in soil on infected plant debris, so it is very important that cover crop plants are not susceptible and pathogen spores do not accumulate in the soil In breeding programmes, together with agronomic and qualitative characteristics of the product it is important to include resistance to the main pests and diseases, thus providing healthier products with environmental and consumer bene ts. The exploitation of new sources of DM disease resistance represents an important strategy in order to improve cultivated radish production. Robust phenotyping data are fundamental for accurate germplasm selection and future use, although few radish genotypes resistant to DM were identi ed so far (Jiang et  Due to the very short production cycle of radish a source of resistance to be effective must cover all the organs of the plant. The objectives of the present study were to develop a screening methodology for assessing DM resistance in different radish plant organs, to use this methodology to identify sources of resistance by screening a germplasm collection, and to compare the expression of resistance in cotyledons, true-leaves and roots.

Material And Methods
Plant material and plantlet production A group of radish accessions with known cotyledon resistance to H. brassicae f. sp. raphani was selected for testing seedling and root resistance ( Table 1). The accessions had different origins, genetic backgrounds (breeding lines, commercial varieties and genebanks), and growing cycles. The radish accession Rd197 was used to obtain fresh H. brassicae inoculum for all experiments and was included in the tests as a susceptible control.

Origin of pathogen isolates and inoculum preparation
The resistance of cotyledons and true-leaves was tested with the H. brassicae isolate R10, and the roots were tested with isolates R10 and R6 in two independent experiments. The H. brassicae isolates were collected in eld plants of Raphanus sativus var. sativus in different geographic origins. The isolate R10 was provided by Syngenta Seeds and was collected in cotyledons in the Netherlands (Venhuizen). The H. brassicae isolate R6 was provided by Gautier Seeds and was collected from roots in France (Bouches du Rhone). Field isolates were isolated, cleaned, and noncontaminated H. brassicae isolates were stored at -18°C in infected cotyledons of the susceptible accession Rd197.
Spore suspensions of the pathogen were prepared to produce inoculum to be used in the different experiments. Infected cotyledons of the susceptible control recently sporulated with H. brassicae were washed with distilled water, mycelial fragments were removed and the conidia were counted to a 50-75 x 10 3 conidia ml − 1 nal spore concentration using a haemocytometer.
DM screening methodology

Cotyledon inoculation
The cotyledons of six-day-old radish plants were inoculated by drop with a fresh conidial suspension of H. brassicae isolate R10, following the methodology described by Coelho and Monteiro (2018). Brie y, the fully expanded cotyledons were inoculated on the adaxial surface by depositing two 10-µl droplets of the inoculum on each lobe of the cotyledon using a micropipette (Fig. 1a). After inoculation, the plants were incubated at 16 ± 1°C in the dark, for 24-h, inside a propagator (RH = 100%) to support infection. Afterwards the plants were placed in a growth chamber during 5 days under the previously described conditions for seedlings production. Six days postinoculation (dpi), the cotyledons were lightly sprayed with distilled water and re-incubated at 16 ± 1°C in the dark, for 24-h, to induce pathogen sporulation. A total of 24 plants per accession were evaluated at cotyledon stage in three independent replications.

Leaf inoculation
The rst two leaves of 14-day-old radish plants were inoculated by pulverization using a handheld sprayer with a fresh conidial suspension of H. brassicae isolate R10 (Fig. 1d). The inoculated plants were submitted to the procedures previously described for cotyledon test, but a longer period for infection was necessary. Following an initial 24-h incubation period, plants were placed in a growth chamber during 10 days and individually scored for H. brassicae infection, after a 24-h incubation period. Two leaves per plant in a total of 10 plants per accession were tested in two independent replications.

Root inoculation
The roots of 14-day-old radish plants were inoculated by pulverization in separate trays with H. brassicae isolates R10 and R6, following the procedures described for leaf inoculation. The radish seeds were seeded super cially in alternate rows in order to facilitate the pulverization of the roots, and the radish root resistance was individually assessed at 12dpi. The isolates were tested in different experiments and a total of 24 plant roots per accession and isolate were evaluated in three independent replications.

Disease assessment and data analysis
The symptoms on cotyledons and on the rst two true-leaves of each plant were evaluated using a visual scale of seven interaction-phenotype classes (IP classes), taking into account the host response and the relative amount of pathogen asexual sporulation (Table 2)   Radish roots were evaluated using a visual scale of ve IP classes (Table 3). Roots classi ed in class 0 indicate no symptoms (immune class) (Fig. 1g); class 1 was a resistant response, showed only necrosis restricted to the point of infection and no sporulation; class 2 was an intermediate response characterized by a rare H. brassicae sporulation con ned to the infection point; and classes 3-4 were susceptible reactions with sparse to abundant sporulation respectively, dispersed over the whole radish surface (Figs. 1h and 1i). Table 3 Interaction-phenotype (IP) classes used to evaluate downy mildew resistance of radish roots. Analysis of variance was performed on the two H. brassicae isolates data at root stage and the signi cant differences between means were identi ed by Tukey HSD test (P ≤ 0.05) using Statistica version 7. The correlations between cotyledon, true-leaf and root DI values were assessed via Pearsonʼs coe cients and the relative P-values signi cance (P < 0.05) were determined.

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Root evaluation with two H. brassicae isolates Unlike the cotyledons and true-leaves that were tested with isolate R10 only, roots were tested with isolates R10 and R6, in two independent experiments. There was a highly signi cant correlation (r = 0.81, P < 0.000) between DI induced by the two isolates (Fig. 2) and most of the accessions showed the same general pattern of resistance when inoculated with each isolate (Table 5).  between isolates in the same accession. Five accessions (Rd001, Rd002, Rd003, Rd004 and Rd130) were resistant at the roots to both isolates and accession Rd198 was highly susceptible to both (Fig. 2).
In Comparing DM resistance in different organs A highly signi cant positive correlation (r = 0.81, P < 0.000) was recorded between DI values of cotyledons and trueleaves (Fig. 3a), but between cotyledons and roots (r = 0.17, P 0.05) and leaves and roots (r = 0.23, P 0.05) the correlation was not signi cant (Figs. 3b and 3c).
Responses were signi cantly different between accession and plant organ concerning resistance/susceptibility to DM. For instance, Rd208 was very susceptible in cotyledons and leaves (DI = 5.6 in both) and showed an interesting partially resistance in the roots (DI = 1.4) to isolate R10. On the contrary, Rd201 was partially resistant in cotyledons and true-leaves (DI = 2.9 and 3.6 respectively) and was susceptible in roots (DI = 2.4). Even more contrasting was the accession Rd198 (breeding line) resistance in cotyledons and leaves (DI = 2.0 and 1.2 respectively) and high susceptibility in roots to isolates R10 and R6 (DI = 3.2 and 3.6 respectively). Likewise, four accessions Rd175, Rd176, Rd194, and Rd195 were resistant in cotyledons and true-leaves (DI between 1.7 and 2.0) and susceptible in roots to both isolates (DI between 2.2 and 3.5).

Discussion
In this study we identi ed 35 radish accessions showing potential resistance to DM at cotyledon, leaves and roots (DI ≤ 4.0 for cotyledon and leaves; and DI ≤ 2.0 for roots) from different sources (19 commercial varieties, 14 breeding lines, one landrace and one advanced cultivar). The resistant group includes landrace Rd111 and the advanced cultivar Rd108 from UKVGB (UK Vegetable Genebank), which were resistant in the cotyledons, and the breeding lines Rd017 and Rd020 from the NordGen (Nordic Genetic Resource Center), which were resistant in the cotyledons and 1st and 2nd leaves producing no sporulation.
Information on sources to DM resistance in radish is scarce. Bonnet and Blanchard (1987) (Coelho and Monteiro 2018). These accessions showed also promising resistant responses at leaves and roots, but no information was available about the genetic control of resistance at these stages.
In the current research we inoculated 14 day-old seedlings showing the two rst leaves full expanded and mature. The good correlation between DM resistance at cotyledons and 1st and 2nd true-leaves allows the use of cotyledon resistance to predict adult-plant resistance. The estimate of leaf resistance based on cotyledon resistance would save time and work. Also, cotyledon resistance allowed to assay a large number of plants and observed interaction phenotype (IP) are stable since tests are conducted under controlled environmental conditions. However, in the case of radish cotyledon resistance has its own value because non-infected cotyledons will act as a barrier to slow disease progression to true-leaves and roots. Tronchuda kale (B. oleracea), is a particular case where resistance at cotyledon and adult-plant stages is under the control of two independent genetic systems and so all combinations between cotyledon and mature plant resistance may occur (Monteiro et al. 2005).
To clarify the genetic control responsible for cotyledon, true-leaf and root resistance in radish, genetic studies must be done in the different organs of the plant. However, cotyledon and young true-leaf resistance in radish have higher horticultural relevance because radish has a very short and quick growing cycle. The disease starts on the cotyledons and then progresses to the leaves and roots. Cotyledon resistance may act as a protective barrier to slow the spread of the disease to the crop. Root damage in radish is important because root is the edible part, but the resistance of canopy is also important since it can protect root infection. DM disease attacks throughout the plant cycle and may kill plants or delay their development leading to a huge crop reduction. A good disease control on the leaves is a key issue for high productivity and quality in radish crop.
Root inoculation by spraying is more di cult than applying the same method to cotyledons and leaves, and may be less effective. Part of the root is covered by soil, which promotes some protection against infection, and root infection may be hampered by the higher di culty of retaining inoculum drops on root surface, in comparison with cotyledons and leaves that have horizontal surfaces. However, the consistency of the results of the two independent root inoculations with isolate R10 and R6 isolates shows that the method we used to test the roots was reliable.
The seven Japanese radish daikon accessions (Raphanus sativus L. var. longipinnatus L.H. Bailey) evaluated in this study have a longer vegetative cycle than the conventional radish varieties, need 50-80 days to harvest (APA 1988), may grow up to 75-cm long with a diameter of up to 25-cm and weigh several kilograms. The roots of these plant were tested at an earlier stage of development in comparison with standard radishes. To con rm whether the stage of development may affect the expression of root resistance, daikon radish should be tested later on during the cycle or ideally under eld conditions. Declarations Figure 2 Correlation between Disease Index (DI) values of thirty-six radish accessions inoculated with H. brassicae isolates R10 and R6 on the roots (r=0.81, P < 0.000). Figure 3