Evaluation of Coffea arabica Cultivars for Resistance to Meloidogyne konaensis

Abstract

Coffee is an important agricultural crop for the State of Hawaii. Developing new coffee cultivars with resistance to damaging pests and diseases is crucial for improving production and maintaining the profitability of the industry. With the recent discovery of Hemileia vastatrix, coffee leaf rust (CLR), to Hawaii there is a strong interest in replanting with CLR-resistant germplasm. However, when selecting an appropriate cultivar for replanting, susceptibility to other pathogens, such as plant-parasitic nematodes, should be taken into consideration. Meloidogyne konaensis, the Kona coffee root-knot nematode, causes severe destruction of the root system, reducing the yield and causing the mortality of susceptible Coffea arabica cv. Typica trees. Fortunately, resistance to root-knot nematodes has been found in some C. arabica germplasm. In this study, accessions of wild Ethiopian C. arabica and two CLR-resistant C. arabica cultivars, Tupi-HI and Obata, were evaluated for resistance to M. konaensis in a greenhouse bioassay. All Ethiopian accessions retained high levels of resistance, resulting in reproductive factors (Rfs) lower than 1.0, and low root-rot ratings. Tupi-HI was highly susceptible to M. konanensis, with an Rf value of 7.12, whereas Obata was slightly susceptible, with an Rf value of 2.33. Both cultivars had high root-rot ratings, suggesting intolerance to the nematode. Hybridizing Ethiopian C. arabica with Tupi-HI or Obata may result in new elite cultivars, resistant to both H. vastatrix and M. konaensis.

1. Introduction

Coffee is the third highest valued agricultural crop in the State of Hawaii, with annual sales of USD 62 million [1] and predicted downstream economic benefits of USD 500 million. Unfortunately, crop yields are in decline due to destructive pests and diseases. Meloidogyne konaensis [2], Kona coffee root-knot nematode, is a devastating pathogen of Coffee arabica cv. Typica, the predominant cultivar grown on Hawai`i Island. Swelling and corkiness of the main tap root occur with M. konaensis infestation, as well as root galling, necrosis, and a reduction in feeder roots [3]. The resulting root damage leads to yellowing and wilting of the leaves, followed by defoliation, stunting, and low yields. In fields with heavy infestations, mortality can transpire within 13 years in 80% of ungrafted Typica trees [4].

Utilizing host–plant resistance is one of the most effective and easily adoptable management techniques for controlling root-knot nematodes. Currently, growers graft susceptible Typica scions to nematode-tolerant rootstocks C. liberica cv. Dewevrei or C. canephora at the hypocotylendon stage, using the Reyna method [5].

Traditional coffee breeding is another alternative for optimizing host–plant resistance to manage M. konaensis and other pathogens. Cultivars resistant to pests and diseases can be hybridized with cultivars with desirable cupping quality and other favorable agronomic characteristics. One important source of resistance is from wild Ethiopian C. arabica accessions, which have exhibited resistance to several root-knot nematode species, including M. konaensis [6], M. paranaensis [7], and M. incognita [8]. Because resistance to pests and pathogens can break down over successive generations, the current study was undertaken to confirm that M. konaensis resistance remained durable in Ethiopian Arabica seeds collected from an open-pollinated field of mixed cultivars.

Another pathogen detrimental to coffee farms in Hawaii is Hemileia vastatrix, coffee leaf rust (CLR). Discovered in 2020 in the Kona region of Hawai`i Island and other coffee-growing areas throughout the State, this fungal pathogen has caused significant production losses. Control of this disease on susceptible coffee varieties requires good plant nutrition, good aeration in the field, and multiple fungicide applications, which add to production costs and reduce farmer profit margins. Although contact copper-based fungicides are available for initial control, host–plant resistance remains the best long-term solution for managing this disease. The C. arabica cultivars Tupi and Obata, developed by the Instituto Agronômico (IAC) of Sao Paulo State in Campinas, Brazil, are known for rust resistance and good cupping quality. Both are progenies of Sarchimor and Catuai crosses, and have been selected over many years. Careful evaluations of these cultivars need to be undertaken, as a high degree of variability in resistance to H. vastatrix, Colletotrichum kahawae, and Meloidogyne spp. has been observed in Sarchimor populations [9]. With growers showing interest in propagating and planting these cultivars, it is crucial that we understand their potential susceptibility to M. konaensis before extensive field plantings are established. In this study, we aimed to test the susceptibility of Tupi and Obata, to learn if a recommendation of grafting these cultivars to nematode-tolerant rootstocks is necessary for maintaining the long-term vitality and longevity of the crop. In a greenhouse bioassay, we determined the levels of resistance and tolerance of the Ethiopian Arabica accessions and the CLR-resistant cultivars to M. konaensis by evaluating nematode reproduction, root-rot ratings, root-gall ratings, root weight, and plant growth.

2. Materials and Methods

Coffee seeds were collected from the Hawaii Agriculture Research Center’s coffee germplasm field in Kunia, Hawaii, and sent to the USDA’s ARS Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center in Hilo, Hawaii for grow-out and screening. Three Ethiopian Arabica accessions, E17 (T.16706-6), E25 (T.16712-1), and E52 (T.16733-2), were evaluated for resistance to M. konaensis. Open pollinated seeds of Ethiopian Arabica were collected from multiple trees for each cultivar. In addition, seeds were harvested from individual trees numbers 3 and 4 from accession E17 (ET17T3 and ET17T4), and tree number 3 from accession ET 25B (ET25BT3), to evaluate for signs of segregation. These were compared with the standards C. arabica cv. Typica as a susceptible control, and C. canephora cv. Nemaya (N204) as a resistant control. The cultivars Tupi and Obata were also included in the trial. Recent genotyping has discovered that the cultivar imported to Hawaii as Tupi did not genetically match up with the known cultivar. Its actual identity has not been confirmed at this time, and it is referred to in this study as Tupi-HI for differentiation from the original cultivar.

Seeds were germinated in 13 cm diameter community pots containing vermiculite. A total of 18 seedlings of the 7 cultivars, plus seedlings originating from three individual trees, were chosen and transplanted into tree pots (10 cm × 24 cm) filled with an autoclaved 50:50 soil–sand mixture. Nutricote^® 240-day slow-release 13-13-13 fertilizer with micronutrients (Chisso–Asahi Fertilizer Co., Ltd, Tokyo, Japan) was applied. Plants were arranged in a completely randomized design in a greenhouse under 73% shade. Day temperatures ranged from 24 to 32 °C, and night temperatures from 14 to 24 °C. When plants were approximately 30 cm in height, half were inoculated with 2500 eggs of M. konaensis, and the remaining were treated with water only. The inoculum was dispensed, using a pipette in a volume of 10 mL aqueous solution, around the collar of the plant, and covered lightly with soil. Plant height was measured at the time of inoculation.

The inoculum originated from the University of Hawaii College of Tropical Agriculture and Human Resources Kainaliu Experiment Station in Kealakekua, Hawaii, using Solanum lycopersicum “Orange Pixie” (tomato) plants as a trap crop. Tomato seedlings were planted into an infested coffee field adjacent to C. arabica trees grafted onto various rootstocks. After 60 days, the tomato plants were removed from the field, and the roots were washed and excised. The roots were blended in a 0.6% sodium hypochlorite, NaOCL solution, and rinsed through a 150 μm pore sieve nested on a 20 μm pore sieve [10]. Nematode eggs and juveniles (J2) were collected in a 50 mL aqueous solution, and a 0.5 mL subsample was counted under a Leica inverted microscope. Eggs were allowed to hatch, and species identification was confirmed using morphological parameters [2].

Twelve months after inoculation, plant height was measured, and the experiment was terminated. Roots were excised, washed, and weighed. Root health ratings were performed using a 0–5 scale, where 0 was defined as good root health, with no visible signs of root rot, and 5 as poor health, with root rot visible on 100% of roots. A gall index was conducted using the Zeck root-knot rating scale [11], with 0 representing no galls or swelling and an abundance of feeder roots, whereas 10 represented galls and swelling on the entire root system, with a lack of feeder roots. After the ratings were conducted, the coffee roots were processed, and nematode eggs and J2 were extracted and counted as described above for the inoculum.

The nematode reproductive factor (Rf) was calculated by dividing the final nematode population by the initial nematode population (Pf/Pi). Resistance to M. konaensis was characterized with an Rf < 1, susceptibility with Rf > 1, and immunity with Rf = 0 [12]. Analysis of variance was conducted on the data, and the means separated by the least significant difference test (JMP, Version 14.2. SAS Institute Inc., Cary, NC, USA, 1989–2021).

3. Results

The Ethiopian Arabica accessions demonstrated high levels of resistance and tolerance to M. konaensis, comparable to the resistant rootstock Nemaya. In contrast, Tupi-HI was most similar to the susceptible and intolerant Typica cultivar. The reproductive factors of Typica and Tupi-HI after 12 months were different from all the other cultivars tested (p < 0.0001) (Figure 1). Both had Rf values between 7 and 8, whereas the remaining cultivars had Rf values below 1.0, except Obata, with an Rf value of 2.33. Evidence of segregation in Obata plants occurred, with Rf values ranging from 0.1 to 10.5. Forty-four percent of the Obata plants evaluated could be considered resistant, with Rf values below 1, although the remaining Obata plants had high nematode populations, and were categorized as susceptible. Tupi-HI also demonstrated a high range of variability, ranging from 0.7 to 17.5, although only one plant fell within the resistant range. Nemaya plants were highly resistant; 78% of plants were considered immune, with no nematode reproduction after 12 months.

A similar trend to that of the Rf value was observed in the total number of eggs and J2 recovered per plant (p < 0.0001). Sixty-seven percent of the susceptible Typica roots produced over 15,000 eggs and J2. The largest population of nematodes (43,868 eggs and juveniles) was recovered from a Tupi-HI root system. In general, Obata roots supported fewer than 5000 eggs and J2 per plant, except for two root systems with large populations over 10,000. The eggs per gram of root were not different between the cultivars (p = 0.4245).

Nemaya and the Ethiopian Arabica accessions had lower root-rot ratings than Obata, Tupi-HI, and Typica (p < 0.0001) (Figure 2). Obata had the highest root-rot rating among all cultivars in the trial, although it did not differ from Tupi-HI (p = 0.2217). A difference in the Zeck gall-rating index between Tupi-HI and all Ethiopian Arabica accessions was observed (p < 0.0001), whereas the gall rating of Tupi-HI was greater than, but still statistically similar to, Typica, Obata, and Nemaya (p > 0.05). The means of most cultivars did not separate with this analysis.

Inoculated Nemaya (19.4 g) and Tupi-HI (18.6 g) had higher root weights than the Ethiopian Arabica accessions (p < 0.0001), whereas inoculated Obata (15.3 g) and Typica (12.1 g) were not different from most of the other plants tested. Overall, root weights were higher in uninoculated plants (p = 0.0418) (13.6 g vs. 10.2 g). Uninoculated plants also had higher growth rates (101%) than plants inoculated with M. konaensis (80%) (p = 0.0115). Inoculated seedlings from tree number 3 of Ethiopian Arabica accession ET17 had the highest plant growth over all inoculated cultivars tested (p < 0.0001). The growth rate of inoculated Typica (104%) was higher than those of inoculated Tupi (63%), Obata (27%), and Nemaya (25%) (p = 0.05).

4. Discussion

The Ethiopian Arabica accessions in this trial maintained the resistance to M. konaensis observed in earlier studies. Anzueto et al. [8] also observed that self-pollinated F1 Ethiopian Arabica hybrids maintained their resistance to root-knot nematodes in the F2 populations. This source of nematode resistance has excellent potential to be used in coffee-breeding programs. Hybridizing these plants with rust-resistant cultivars could lead to the development of an elite cultivar with nematode and rust resistance, allowing Hawai`i coffee growers to control the two most devastating pathogens in their production systems using one easily adoptable methodology.

Due to the observed results, our recommendation for growers interested in immediately replanting their fields with the rust-resistant cultivars Tupi-HI and Obata is to graft them on nematode-tolerant rootstocks. The susceptibility and intolerance of Tupi-HI and Obata to M. konaensis could result in poor plant growth, low yields, and mortality over time, as the disease from nematode infestation progresses. In a 13 year field study conducted in an M. konaensis-infested plot, susceptible Typica grafted on nematode-tolerant C. liberica cv. Arnoldiana had no mortality, and the highest cherry yields compared to other rootstocks and ungrafted Typica trees [4]. Typica grafted on C. liberica cv. Dewevrei and C. canephora cv. Nemaya also had low mortality and high yields compared to ungrafted controls.

The reproductive factor was the most effective means of characterizing resistance or susceptibility in the cultivars tested, whereas the best indicator of assessing nematode tolerance and susceptibility among the cultivars was the root-rot rating. Developing an effective visual rating methodology for determining resistance and tolerance would prevent a destructive assay, and allow characterized plants to be used within the breeding program. Rezende et al. [13] found that the gall index and thickness (GIT) rating was positively correlated with the reproductive factor, and recommended its use in coffee-breeding programs. However, the Zeck root-knot nematode rating scale used in this study did not demonstrate the same results. This is likely due to the species-specific behavior of M. konaensis to infest and reside in the tap root, instead of finer feeder roots where galls can be visualized and counted.

Plant height was also not a good indicator of tolerance, due to the varying heights of plants at the onset of the trial. Smaller plants, such as the Ethiopian Arabica accessions, demonstrated higher gains in growth than the cultivars that began the trial as larger plants. Surprisingly, in this study, Nemaya plants had low vegetative growth rates. In previous trials, Nemaya had a vigorous growth rate in the presence of M. konaensis, superior to Typica in potted plants [14] and when used as a rootstock in infested fields [4]. Nemaya plants did, however, have the highest root weights, and little or no nematode reproduction. The root-rot rating was low in Nemaya, but moderate for the root gall index. Ethiopian Arabica accessions, while highly resistant, also had the lowest root weights.

Future research involves screening CLR-resistant cultivars imported from other coffee-growing countries for their resistance and tolerance to M. konanensis. Management strategies, such as the use of nematode-tolerant or -resistant rootstocks, are crucial for maintaining high productivity and longevity in newly-replanted coffee plantations. Until new cultivars are developed and evaluated for both CLR and root-knot nematode resistance, or only CLR-resistant lines are available, grafting is a viable control strategy against M. konaensis. Long-term goals include developing commercial cultivars with specialty coffee cupping quality, favorable agronomic traits, and resistance genes against root-knot nematodes and coffee leaf rust.