Evaluation of Loofah Lines for Resistance to Tomato Leaf Curl New Delhi Virus and Downy Mildew, as well as Key Horticultural Traits

Two loofah (Luffa) species, the ridge gourd (Luffa acutangula (L.) Roxb.) and the sponge gourd (L. cylindrica (L.) M. Roem.; syn. L. aegyptiaca), are cultivated widely in Asia by smallholder famers. Both species have significant economic and nutritional importance. However, Tomato leaf curl New Delhi virus (ToLCNDV) and downy mildew (DM) caused by Pseudoperonospora cubensis are important biotic constraints to loofah production throughout Asia. Loofah landrace-derived breeding lines, developed at the World Vegetable Center (WorldVeg), were evaluated at the WorldVeg East and Southeast Asia Research and Training Station, Kasetsart University, Kamphaeng Saen, Thailand—where natural epidemics of ToLCNDV and DM regularly occur. The lines were also evaluated for other commercially important horticultural traits such as days to 50% staminate and pistillate flowering, fruit color, fruit bitterness, and market segment classification. Thirteen and 59 lines of ridge gourd and sponge gourd, respectively, were determined to be resistant to both ToLCNDV and DM. These lines covered all market segments of loofah and exhibited variability for all of the evaluated horticultural traits. The results of these evaluations and their implications on loofah breeding are discussed.


Introduction
Ridge gourd (L. acutangula (L.) Roxb.) and sponge gourd (Luffa cylindrica (L.) M. Roem.; syn. L. aegyptiaca Mill.) are the principal cultivated species in the genus Luffa [1]. Seven additional wild species of Luffa include L. graveolens Roxb. (var. longistyla), L. echinata Roxb., L. tuberosa Roxb., L. umbellata Roem, L. quinquefda (Hook and Arn), L. astorii Svans, and L. saccata. In Asia, the two cultivated species (hereafter referred to as loofah) are a commercially and nutritionally significant cucurbitaceous market vegetable. Immature loofah fruit have a significant role in Asian cuisines and are eaten boiled, peeled, and fried, and in curries and soups. Loofah fruits contribute calcium (20 mg/100 g Fresh Weight (FW), magnesium (14 mg/100 g FW), potassium (139 mg/100 g FW), and vitamin A (410 IU) to the human diet [2]. A 200 g serving of loofah fruit provides between 5% and 16% of the daily recommended intake, respectively, of the previously noted nutrients. In addition to their DM resistance. Here, we report the results of the field screening of the WorldVeg loofah breeding lines (S 5 generation) for their reaction to ToLCNDV and DM, and their evaluation for key horticultural traits.

Materials and Methods
A field screening (two growing seasons per year) of the WorldVeg loofah germplasm collection of landraces consisting of 467 accessions of ridge gourd and 783 accessions of sponge gourd, collected from 10 Asian countries against ToLCNDV and DM, began in 2016 at the WorldVeg East and Southeast Asia Research and Training Station, Kasetsart University, Kamphaeng Saen, Thailand. Populations were initially segregated for resistance to ToLCNDV and DM, and for fruit traits such as color, shape, length, bitterness, and duration of fruit harvest. Plants tolerant to ToLCNDV and DM and belonging to different market segments (bitter vs. non-bitter fruit), were selected and hand-pollinated (selfed) to produce progeny, which were used in the subsequent growing/selection cycles (plant to progeny row). Six plants of each ridge gourd (82) and sponge gourd (65) breeding line (Tables 1 and 2), developed through this breeding approach (S 5 generation), and a susceptible check for both ToLCNDV and DM (20 plants each), were transplanted into single 9.6 m 2 plots on 25 December 2019. Natural epidemics of ToLCNDV and DM occurred on the loofah during this period.
Plants were rated in the field 60 days after transplanting for ToLCNDV severity using a 0-2 scale: 0 = no visible symptoms, 1 = mild symptoms (slight chlorosis of leaves), and 2 = severe symptoms (severe chlorosis of leaves, upward leaf curling, and stunting of plants). Severity ratings of 0, 1, and 2 corresponded to resistant, moderately resistant, and susceptible, respectively. To confirm the presence of ToLCNDV, diseased leaf tissue from individual plants of the susceptible checks were crushed in a 500 µL Tris-Ethylenediaminetetraacetic acid (TE) buffer, and DNA was extracted as described previously [34]. The quality of the DNA was evaluated on a 1% agarose gel and stored at −20 • C until further use. PCR amplifications were performed using a ToLCNDV-specifc primer pair Beg434F + ToLC1524R (developed by Dr. Orawan Chatchawankanphanich, The National Science and Technology Development Agency (NSTDA), Kasetsart University, Thailand). DNA fragments of the expected size, ca. 1.254 bp, were amplified. All of the samples of the control plants collected from the loofah experimental plot were positive, as per the PCR analysis.
DM severity was also assessed 60 days after transplanting. The genotypes were categorized into three groups, namely, immune (0%), resistant (1-10%), moderately resistant (11-30%), and susceptible (>30%), based on the percentage of symptomatic leaf area (angular lesions), as determined using a modified procedure of that described previously [35]. The presence of Pseudoperonospora cubensis was confirmed based on the culture morphology and microscopic examination of sporangiophores and sporangia as described by Waterhouse and Brothers [36].
The following horticultural traits were recorded: (1) days to 50% pistillate flowering after transplanting, (2) days to 50% staminate flowering after transplanting, (3) fruit color, (4) market segment of ridge gourd based on fruit length (short (up to 30 cm), medium (31-44 cm), and long (>45 cm)), and (5) market segment based on fruit length in sponge gourd (short (up to 20 cm), medium (21-30 cm), and long (>30 cm)). The number of days from transplanting to the first open pistillate and staminate flowers was recorded for each plant. Fruit bitterness was determined using three fresh fruits of each line that were harvested at marketable maturity. These were washed and cut into small (ca. 3 g) pieces (after removing the terminal three inches of each fruit) and used for organoleptic assessment by a three-person taste panel. Two categories of bitterness were recorded-non-bitter and bitter. The evaluators rinsed their mouths with water after each sample tasting.

Evaluation of Luffa acutangula Lines
"THLA 456" (susceptible check) was susceptible to ToLCNDV (mean rating = 2) and DM (>41% symptomatic leaf area). Eighty-two lines of Luffa acutangula evaluated in this study were resistant to ToLCNDV (mean rating = 0; Table 1). A majority (62/76.8%) of the ToLCNDV-resistant lines were derived from landraces originating in Bangladesh, while 20 (24.1%) of the resistant lines were developed from the landraces originating in Thailand. A single virus-resistant line originated in the Philippines. These ToLCNDV-resistant lines were categorized into three commercial market segments based on fruit length, namely: short (47/57.3%), medium (30/36.6%), and long (5/6.1%; Figure 1). The fruits of these lines were nonbitter. Three distinct fruit skin colors were observed in the ToLCNDV-resistant lines, namely: green (75/91.4%), light green (1/1.2%), and dark green (6/7.4%). Days to 50% pistillate and staminate flowering (after transplanting) ranged from 21-50 and 21-46, respectively, among these ToLCNDV-resistant lines. The earliest (<25 days to 50% flowering) pistillate flowering lines originated in Bangladesh. Resistance to ToLCNDV in ridge gourd is conditioned by a single dominant gene [37]. A dark green fruit color is dominant over a green and light green color, whereas a green fruit color is dominant over light Resistance to ToLCNDV in ridge gourd is conditioned by a single dominant gene [37]. A dark green fruit color is dominant over a green and light green color, whereas a green fruit color is dominant over light green [37]. The identified ToLCNDV-resistant lines provide ample opportunity for the development of early maturing, virus-resistant F 1 hybrid cultivars of various market segments possessing the dark green and green fruit colors preferred by consumers.
Six and three DM-resistant lines originated in Bangladesh and Thailand, respectively. All were non-bitter. The fruit colors of eight and one DM-resistant lines were green and dark green, respectively. Ridge gourd cultivars resistant to DM are not currently available. Hence, this germplasm is a potential source of resistance for DM in ridge gourd and is of importance to public and private sector breeding programs in Asia.
green [37]. The identified ToLCNDV-resistant lines provide ample opportunity for the development of early maturing, virus-resistant F1 hybrid cultivars of various market segments possessing the dark green and green fruit colors preferred by consumers.
Six and three DM-resistant lines originated in Bangladesh and Thailand, respectively. All were nonbitter. The fruit colors of eight and one DM-resistant lines were green and dark green, respectively. Ridge gourd cultivars resistant to DM are not currently available. Hence, this germplasm is a potential source of resistance for DM in ridge gourd and is of importance to public and private sector breeding programs in Asia.
Although none of the sponge gourd lines were immune to DM, 23 lines were resistant (1%-10% symptomatic leaf area). Resistant lines (36) originated in Bangladesh and Southeast Asia (22). Sponge gourd Resistance to ToLCNDV in sponge gourd was determined to be governed by a single dominant gene [38]. Commercial sponge gourd cultivars resistant to ToLCNDV are currently unavailable in the global seed market. Therefore, the ToLCNDV-resistant lines described here provide an opportunity for the development of virus resistant F 1 hybrid cultivars.
Although none of the sponge gourd lines were immune to DM, 23 lines were resistant (1-10% symptomatic leaf area). Resistant lines (36) originated in Bangladesh and Southeast Asia (22). Sponge gourd cultivars resistant to DM are unavailable in all sponge gourd market segments. Hence, this germplasm could be of use in the development of DM-resistant cultivars, so as to fill the various market segments.

Discussion
ToLCNDV infecting cucurbits results in major economic losses among cucurbits, including loofah, in Asia. The disease can result in 100% crop loss under epidemic conditions [39][40][41]. A sponge gourd accession "DSG-6" from India was previously reported to be resistant to ToLCNDV. That resistance was determined to be governed by a single dominant gene [38]. However, the seed industry has not been able to exploit that resistance to develop F 1 hybrids, as the resistance failed to persist during multi-location trials across India [37]. In addition, the fruits of "DSG-6" are dark green with superficial stripes. However, the sponge gourd market in India, Pakistan, and Bangladesh is dominated by cultivars producing green or light green fruit without stripes. "DSG-6" originated in West Bengal (India). Interestingly, the majority of the sponge gourd lines found to be resistant to ToLCNDV in this study originated in Bangladesh, which was part of the West Bengal state of India before the partition of British India in 1947. The Indo-Burma region and India (including Bangladesh) are considered the primary centers of diversity of sponge gourd and ridge gourd, respectively [42], although the exact area of domestication of sponge and ridge gourds is disputed [43]. Sponge gourd material from Bangladesh also provided the earliest pistillate flowering lines. Ridge gourd lines resistant to ToLCNDV have not been previously reported, although Premchand et al. [44] evaluated germplasm from India for resistance to this begomovirus. The first commercial ridge gourd F 1 hybrid cultivar "Arti", resistant to ToLCNDV, was released in India by VNR Seeds Private Limited in 2008 [37]. However, the resistance proved to be unstable across locations in India.
Downy mildew was observed in sponge gourd fields of commercial farmers in the Odisha state of India in 2014 [24]. Sources of DM resistance in ridge gourd and sponge gourd have not been reported. The current study has identified sources of DM resistance in the short and medium fruit length market segments of ridge gourd, and in the short, medium, and long fruit length market segments of sponge gourd. These include green, light green, and dark green fruit skin colors. These resistant lines provide a basis for the development of new DM-resistant elite ridge gourd and sponge gourd F 1 hybrids. Multiple genes for DM resistance have been reported in cucumber and melon [45,46]. Information on the genetic mechanisms of resistance to DM in loofah is lacking in the literature.
ToLCNDV-and DM-resistant lines will be showcased during the WorldVeg's Loofah Open Field Day in 2021. These lines are being further evaluated for ToLCNDV and DM in multi-location trials across Asia, in partnership with seed companies and public institutes. SNP-based genetic diversity analysis of these lines, and a study of their relationship with commercial hybrid cultivars currently popular with the farmers in Asia, is in progress.

Conclusions
Ridge gourd and sponge gourd fruit contribute to human nutrition, and their production and sale provides a livelihood for resource-poor farmers in Asia. These cucurbits are also important members of home, school, and community gardens in the tropics. ToLCNDV and DM are the major production constraints faced by growers in Asia, and commercial cultivars resistant to both these economic diseases are currently unavailable. The adoption of disease-resistant cultivars is an economically sound approach for disease management. The very few currently available hybrid cultivars of loofah are popular with farmers. However, these cultivars have a narrow genetic base. The current work utilized the genetically diverse landraces of loofah maintained in the WorldVeg genebank to identify and develop loofah inbred lines resistant to ToLCNDV and DM. This assortment of genetically broad-based breeding lines includes those producing fruits displaying horticultural traits associated with various commercial segments of loofah. Hence, these lines provide an opportunity for the global loofah seed industry to develop new F 1 hybrids resistant to ToLCNDV and DM to meet the needs of both growers and consumers. The majority of the loofah lines resistant to ToLCNDV and DM originated in Bangladesh. Thus, future loofah germplasm collection and conservation efforts should focus on the loofah landraces present in the different agro-climatic regions of that country.