Host status of melon, carrot, and Meloidogyne incognita-susceptible and -resistant cotton, cowpea, pepper, and tomato for M. floridensis from California

Meloidogyne isolates: The isolation, culturing, and identification of the California Meloidogyne floridensis isolate (Mflor) and the California M. incognita race-3 isolate (Minc-3) used in this study was as described previously (Ploeg et al., 2023; Ploeg and Stoddard, 2023). In short, M. floridensis was isolated from infected galled almond rootstock, and a single-egg-mass isolate was multiplied and cultured on tomato Daniela plants in a greenhouse inside the Nematode Quarantine Facility at the University of California Riverside under CDFA permit 398. The greenhouse temperature ranged between 21–26 °C, and plants were grown under natural light and fertilized with 5 g of Osmocote 17–6–10 controlled release fertilizer once every 8 weeks (Scotts-Sierra Horticultural Products Co, Marysville, OH). Species identification was done by PCR (Westphal et al., 2019) using second-stage juveniles that were hatched from the egg suspension used as inoculum. The M. incognita race-3 isolate (Minc-3) was originally isolated from cotton in the San Joaquin Valley, California, and has been maintained on susceptible tomatoes in our greenhouse for several years. Species identification was confirmed through PCR as described by Dong et al. (2001) using primers FMi 5′-CTC TGC CCA ATG AGC TGT CC-3′ and RMi 5′-CTCTGCCCTCACATTAAG-3′. Eggs of Mflor and Minc-3 used as inoculum in the experiments were extracted from roots of tomato Daniela by shaking for 3 min in a 0.5% NaOCl solution (Hussey and Barker, 1973) and collected by washing over two stacked 25 μm pore-size sieves.

RKN reproduction on selected crop cultivars: Root-galling and multiplication of Mflor and Minc-3 on M. incognita-susceptible tomato cultivar Daniela (Osborne Quality Seeds, Mt. Vernon, WA), pepper cultivar Baron (Seminis, Oxnard, CA), cotton cultivar SJ2 (originally obtained from California Planting Cotton Seed Distributors and propagated in our greenhouse), cowpea cultivar CB46 Null-1 (seed kindly provided by Dr. Huynh, Dept. Nematology, UC Riverside), carrot cultivar Imperator 58 (Mountain Valley Seed, Salt Lake City, UT), and melon cultivar Durango (Seminis, Oxnard, CA) and on M. incognita-resistant tomato cultivar Celebrity (Bayer/Seminis, St. Louis, MO), pepper cultivar Carolina Wonder (Reimer Seeds, Saint Leonard, MD), cotton cultivar DP 1747NR B2XF (seed kindly provided by Dr. Wheeler, Texas A&M), and cowpea cultivar CB46 (seed kindly supplied by Dr. Huynh, Dept. Nematology, UC Riverside) was determined in a replicated quarantine-greenhouse pot study. Tomato and pepper were seeded in a multicell seedling tray with potting mix (Sunshine Mix 5, Sungro, Vancouver, BC, Canada) and placed in a greenhouse. Three (tomato) or four (pepper) weeks after emergence, plants were carefully removed from the seedling trays, and roots gently washed free of potting mix and transplanted into 1-liter white plastic cups filled with steam-sterilized sand (93% sand, 4% silt, 3% clay, pH 7.1). Cotton, carrot, cowpea, and melon were seeded directly in 1-liter white plastic cups filled with steam-sterilized sand (three seeds per cup) and thinned to leave one plant per cup one week after emergence. Ten pots were set up for each crop cultivar, and five pots were each inoculated with 10 ml of a suspension containing 1,000 eggs/ml of Mflor and five pots with Minc-3 by adding 2.5 ml to each of four holes around the roots of each plant (10,000 eggs per plant). All pots were inoculated at the same time (tomato and pepper 1 week after transplanting, carrot 4 weeks after emergence, cotton 3 weeks after emergence, cowpea and melon 2 weeks after emergence). The pots were randomized over a metal galvanized greenhouse bench, allowing free water draining. They were watered daily with a slow drip to avoid water/soil splashing contamination among pots through an automated drip-irrigation system and fertilized one week after inoculation with 5 g of Osmocote 17–6–10 controlled release fertilizer (Scotts-Sierra Horticultural Products Co, Marysville, OH). The greenhouse temperature ranged between 21–26 °C under natural light. Plants were removed after 8 weeks, and the roots were washed free of soil. They were rated for the presence of root galling (0–10 scale: 0=no galling, 10=100% of root system galled. Bridge and Page, 1980). Eggs were extracted from the roots by shaking for 3 min in a 0.5% NaOCl solution (Hussey and Barker, 1973), collected by washing over two stacked 25 μm pore-size sieves, and counted under a dissecting microscope at 40x magnification. The complete experiment was repeated once.

The effect of the two RKN isolates on root-galling and egg production was analyzed separately for each plant cultivar. Prior to analysis, egg counts were transformed by x¹ = log₁₀(x+1) to standardize the variance. The significance of the RKN isolate, the repeated experiment (1 or 2), and the interactive effect on egg production was determined using the ANOVA procedure in SAS statistical software. Where treatment effects were significant at the 95% confidence level, treatment means were compared using Fisher's LSD procedure also at the 95% confidence level. Effects on the average galling index were analyzed with the non-parametric Kruskall-Wallis test (SAS Institute, Cary, N.C.).

The interactive effect of “experiment × RKN isolate” on the degree of root galling was significant (P < 0.05) for melon, the susceptible tomato, the resistant pepper, and the susceptible and resistant cowpea. Therefore, the results are shown separately for the two experiments (Table 2).

Roots of melon Durango and the susceptible tomato Daniela were heavily galled, and roots of the resistant cowpea CB46RR remained virtually free of galling after inoculation with either Meloidogyne isolate. Mflor resulted in consistently higher root galling than Minc-3 on the resistant tomato Celebrity and the resistant pepper Carolina Wonder. However, the reverse was true with Minc-3 on the susceptible pepper Baron, resulting in more severe galling than Mflor.

Statistical analysis of the nematode reproduction x¹ = [log₁₀(eggs per root system + 1)] showed that the “experiment x RKN isolate” interactive effect was only significant on the susceptible cowpea CB46Null (results not shown). However, as the separation of the means was the same between the two experiments, the results are presented as the average of 10 replicates.

The host status of a crop cultivar for RKN can be categorized according to the RF of the nematode on that plant as a good host (RF ≥ 1), a poor host (0.1 < RF <1), or a non-host (RF ≤ 0.1) (Oostenbrink, 1966; Sasser et al., 1984). The reproduction factor RF was significantly different between Mflor and Minc-3 on each of the tested crop cultivars (Table 3). On the susceptible crop cultivars tomato Daniela, cowpea CB46Null, pepper Baron, and melon Durango, RF values for Minc-3 were significantly higher than for Mflor, but as RF values on all these crop cultivars were much greater than 1 (RF values: 6.3 – 57.2), they can be considered as good hosts for both RKN isolates (Table 3). The resistance of cowpea CB46RR, pepper Carolina Wonder, and tomato Celebrity to Minc-3 was very strong, with RF values of 0.1, 0.1, and 0.15, respectively. However, Mflor was able to overcome the resistance in tomato and pepper, instead increasing 1.8-fold and 8.2-fold, respectively, on these resistant crop cultivars (Table 3). The resistance in cowpea to Mflor was slightly compromised, and population levels on this crop stayed approximately even over the duration of the trial (RF = 0.9). Both cotton SJ2 and carrot Imperator 58 were good hosts for Minc-3 but poor hosts for Mflor (Table 3). Surprisingly, this difference was not reflected in root-galling (Table 2).

This is the first report on the host status of carrot and melon for M. floridensis. Carrot has been reported in many studies as a good host for four common RKN species: M. incognita, M. javanica, M. arenaria, and M. hapla (Ferris, 2023). Race 1 isolates of the economically important Columbia RKN M. chitwoodi reproduce on carrots and can be distinguished from race 2 isolates, which do not reproduce on this crop (Mojtahedi et al., 1988; Santo et al., 1988). In this study, carrot was a poorer host for M. floridensis (RF = 0.7) than for M. incognita (RF = 3.6). According to these RF values, carrot is designated as a poor host for M. floridensis and as a good host for M. incognita. Whether large differences exist in the host status of carrots for different isolates of M. floridensis remains to be studied. Interestingly, the ability to induce galling on carrots was not different between M. floridensis and M. incognita. Therefore, it is likely that growing carrots on fields infested with M. floridensis will impact carrot quality similarly to M. incognita. Like carrot, melon is also a good host for the most common RKN species (Ferris, 2023). In California, M. incognita is the most important RKN species on melon, resulting in stunting, yield loss, and severe root galling (Radewald et al., 1975). In this study, melon was an excellent host for both M. incognita and M. floridensis, although reproduction of the former was higher (RF 18.0 vs. RF = 9.5). Melon roots were severely galled after inoculation with either RKN isolate. Results on tomato corresponded with our previous results (Ploeg and Stoddard, 2023). The susceptible Daniela is an excellent host for both RKN isolates and the Mi-gene resistant Celebrity is resistant to M. incognita but susceptible to M. floridensis. The ability of M. floridensis to break Mi-gene resistance in tomato also agrees with previous results (Stanley et al., 2009; Marquez and Hajihassani, 2023). Tomato root-galling caused by M. floridensis was light but still significantly higher than after M. incognita inoculation. It can, therefore, be concluded that Mi-gene-carrying tomato cultivars allow the reproduction of M. floridensis and that attempts to use host plant resistance as a management strategy to lower M. floridensis population levels will probably prove ineffective in tomatoes. Cotton SJ2 was a good host for the M. incognita isolate used in this study. This was expected as the isolate was a race 3, able to reproduce on cotton (Taylor and Sasser, 1978). Handoo et al. (2004) reported that cotton was slightly infected and rated it a poor host for M. floridensis. This agrees with our results, as cotton was infested, and the roots developed slight galling, but the population declined (RF = 0.7). The resistant cotton DP 1747NR B2XF was resistant to M. incognita and highly resistant to M. floridensis, and using RKN-resistant cotton promises to be an effective method for lowering both M. incognita and M. floridensis populations. The susceptible cowpea CB46Null was reported as a good host for M. incognita and M. javanica (Ndeve et al., 2018). It was an excellent host for both RKN isolates in our study, resulting in a 57-fold and 37-fold increase in nematode levels for M. incognita and M. floridensis, respectively. The designation of cowpea as a good host for M. floridensis by Marquez and Hajihassani (2023) is confirmed by our results. The previously established strong resistance of the Rk-gene-carrying cowpea CB46 to M. incognita (Ndeve et al., 2018; Ploeg et al., 2023) agrees with our results. However, the resistance of this cowpea cultivar was partially overcome by M. floridensis, which infected but did not increase (RF = 0.9). It caused very minor root-galling on this cultivar, which should be considered a poor host for M. floridensis. Examples of very minimal root-galling coinciding with RKN reproduction on cowpea and Lima beans were observed previously by Ndeve et al. (2018) and Roberts et al. (2008). They determined that in these crops, different genetic factors were involved in resistance to galling and nematode reproduction. It remains to be determined if a similar mechanism can explain the response of cowpea CB46 to M. floridensis. Pepper Baron was a good host for both isolates, although the reproduction of M. incognita was much higher than that of M. floridensis. Galling of the pepper roots was not very strong, even after inoculation with M. incognita, which reached very high egg densities. Previous reports on the host status of pepper for M. floridensis are contradictory, with some studies concluding that pepper was a non-host (Handoo et al., 2004; Kokalis-Burelle and Nyczepir, 2004), while others reported pepper as a host (Stanley et al., 2009). Variability between isolates of M. floridensis or differences in experimental conditions or between pepper cultivars used may be the reason for the reported differences in the host status of pepper for M. floridensis. The N-gene resistance in pepper Carolina Wonder was very effective against M. incognita but ineffective against M. floridensis. Stanley et al. (2009) reported RF values ranging between 0.3 and 1.2 of four M. floridensis isolates from Florida on the N-gene resistant pepper Charleston Belle. They concluded that this pepper cultivar was susceptible to two and resistant to two isolates but that even the two poorly reproducing isolates were able to overcome the N-gene resistance. Compared to their results, the results from our experiments are much less ambiguous as the N-gene-carrying Carolina Wonder can be designated as a good host for M. floridensis.

Interestingly, the reproduction of M. incognita was significantly higher than that of M. floridensis on all the susceptible crop cultivars. This would imply that, under temperatures similar to those in our experiments, a field population consisting of a mixture of M. incognita and M. floridensis under a susceptible crop may, over time, shift to a larger proportion of the population belonging to M. incognita. In our study, pepper Carolina Wonder was a suitable host to distinguish M. incognita from M. floridensis. Other hosts that consistently separated M. floridensis from M. incognita include several M. incognita-resistant Prunus rootstocks such as NemaGuard, NemaRed, FlordaGuard, Guardian, Hansen 536, and Bright's Hybrid 5 (Handoo et al., 2004; Stanley et al., 2009; Westphal et al., 2019).

In conclusion, our results show that M. floridensis is able to overcome resistance in pepper, tomato, and, to a lesser extent, cowpea and that some important California vegetable crops are a host for this nematode (Table 4).

The nematode is relevant to California vegetable production, as it limits resistance-based management options, and consequently, continuing and future efforts to contain, eradicate, and limit the further distribution or new introductions of this nematode are important.