Reaction of Cotton Genotypes to Meloidogyne Incognita Race 3 and Selection Osolates With High Reproductive Rate in Cotton

Background: Among the pathogens that reduce cotton productivity in Brazil, Meloidogyne incognita is one of the most important as it causes severe yield losses and is widespread. The most recommended methods for control of this species are the use of resistant cultivars and crop rotation systems. In Brazil, M. incognita races 3 and 4 have already been reported as cotton parasites but the race 3 is the most commonly found and widely disseminated.This work aimed at selecting virulent and aggressive populations of M. incognita race 3 for testing sources of resistance to this nematode in cotton genotypes. Results: The three isolates of M. incognita race 3 were important for screening of resistant cotton genotypes. The isolate from Umuarama was the most aggressive followed by Moreira Sales and Iporã. The genotypes CD 05-419, CD 05-945, CD 05-1087 and CD 05-1170 showed good performance against M. incognita race 3 in both greenhouse and eld conditions. Conclusions: The cotton genotypes CD05-945, CD05-1170, CD05-1087 and CD05-419 will be selected for future work involving tests for resistance against other important cotton pathogens. Those genotypes can also be used as source of genes for resistance to nematodes in cotton breeding programs. The search for virulent and aggressive M. incognita isolates was very important when screening for resistance in cotton germplasm.


Background
The herbaceous cotton (Gossypium hirsutum L.) is one of the most important annual crops in Brazil, due to its excellent economic pro t and market competitiveness (Dohlman et al., 2019) . In the crop year 2016/2017 Brazil was ranked as the third major exporter and fth largest cotton producer (FAO, 2019). Most of the Brazilian production is concentrated in the states of Mato Grosso do Sul, Goiás, Bahia and Minas Gerais (Silva et al. 2004).
Although cotton producing areas in Brazil have reached high productivity, cotton output has varied in recent years mainly due to the incidence of several pathogens. According to Lawrence et al. , 2014, the losses caused by cotton pathogens are estimated at nearly 12%. However, in areas where root-knot nematodes are present yield losses might be similar or worse than those caused by other cotton pathogens (Blasingame and Patel, 2013).
One of the major nematode species of the cotton crop is the southern root-knot nematode Meloidogyne incognita (Lu et al. 2014). This nematode is highly important due to its worldwide distribution and large host-range. The main symptoms caused by this nematode in cotton plants are root galls and stunting which leads to a decrease in crop yield. The losses in cotton plantations are dependent on levels of this nematode in the soil (Starr and Page, 1990).
M. incognita presents four physiological races but only races 3 and 4 are known to parasitize cotton worldwide (Starr and Page, 1990) with race 3 being the most widespread in commercial areas of cotton production in Brazil (Pires et al. 2008).
The di culties associated with effective control of M. incognita in commercial areas of cotton cultivation are mainly related to the extension of cultivated areas and improper crop management, this latter factor being the most signi cant, as farmers fail to rotate crops seeking immediate return from the land. This leads to a sharp increase in the density of the nematodes which soon become epidemic (Starr et al. 2007).
In cotton growing areas the chemical control of M. incognita may be performed through the use of nematicides. However, these are highly toxic and harmful to the environment, and also increase the costs of cotton production (Overstreet et al. 2014). Furthermore, prolonged use of chemical molecules can foster the selection of resistant variants in the nematode population.
For this reason, the use of cotton resistant varieties is the most desirable method for controlling M. incognita due to its large range of host plants which makes crop rotation a di cult task. In this work, we tested the reaction of several cotton genotypes against different populations of M. incognita race 3, aiming to select sources of resistance to this nematode.

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Collection, establishment and identi cation of M. incognita populations The ve populations of Meloidogyne incognita (EST I1, race 3) used in this study were previously collected in commercial cotton planting areas in Paraná state, Brazil and kept in glasshouse at 25 o C. Meloidogyne incognita populations were identi ed by the esterase phenotype (Esbenshade and Triantaphyllou, 1990) and physiological races were determined (Pires et al. 2008), according to Hartman and Sasser (1985). Single egg masses were extracted from cotton roots and inoculated on tomato plants cv. Rutgers for nematode reproduction. Tomato plants were grown in pots of 1.5 Kg with sterile substrate (1:1 v/v soil and sand) and kept in a greenhouse with humidity control and temperature ranging from 25 to 28 o C.
Extraction of eggs and J2 of M. incognita from tomato roots Sixty days after inoculation (dai) tomato roots were separated from the shoot, washed with tap water, sectioned into small pieces and crushed in a blender with sodium hypochlorite (NaOCl) at 0.5% for 60 seconds and under low rotation (Hussey and Barker, 1973). The suspension was passed through a set of sieves of 60 and 500 mesh. Eggs and J2 retained on the 500 mesh sieve were transferred to a beaker and quanti ed in a Peters' slide before inoculation.

Selection of M. incognita populations for resistance tests in cotton germplasm
Five populations of M. incognita race 3 were tested for virulence and aggressiveness on the resistant cotton varieties IAC 24 and CD 201 and the susceptible FM966. This assay aimed at selecting the most aggressive and virulent populations of M. incognita for testing the cotton germplasm provided. Cotton plants were inoculated with 5,000 eggs of M. incognita and kept in a glasshouse at 25 -28 o C. Cotton plants were assessed at 120 days after inoculation based on the variables gall index, total eggs and reproduction factor.

Cotton genotypes
Twenty eight cotton genotypes, developed by the Central Cooperative of Agricultural Research (Cooedetec) in collaboration with CIRAD-France, were tested against three populations of M. incognita (Table 3). The IAC 24 and FM966 were used as resistant and susceptible cotton cultivars, respectively. The cultivar FMT 701, tolerant to M. incognita, was also included in eld and greenhouse trials together with all the genotypes and the other cultivars.
Greenhouse experiment -Inoculation of M. incognita race 3 on cotton genotypes Cotton genotypes (Table 1) were kept in a greenhouse at a temperature of 27°C and 60% relative humidity. Single cotton plants were grown in plastic tubes of 7 x 18 cm containing sterile substrate composed of soil and sand in the ratio 1.5:1 and fertilized with 2 grams of N-P-K 8-20-20. Plants with two true leaves were inoculated with 3 mL of solution containing 5,000 eggs and juveniles of M. incognita race 3. The quanti cation of eggs and juveniles was determined in a Peters' slide using a light microscope. Water was sprayed on cotton plants twice a day during the experiment. The experimental design was completely randomized with 31 treatments (cotton genotypes and varieties) and ten replicates. Evaluation of greenhouse experiment Cotton genotypes were evaluated at 120 days after inoculation with M. incognita through the following variables: gall index, total eggs and reproduction factor (RF) (Shepherd 1979). Data obtained for eggs were transformed to log X + 1. Reproduction Factor was calculated using the equation RF=Fp/Ip, where Fp means nal population and Ip means initial population. Cotton genotypes with RF values<1.0 were classi ed as resistant (R), Moderately resistant (MR) when 1≥RF<2, Moderately susceptible (MS) with 2≥FR<3 and susceptible (S) when RF>3. This classi cation followed Khan et al. (2016).

Field experiment
Cotton genotypes were cultivated in a commercial area of 120.75 m 2 infested with M. incognita race 3 where cotton had been cultivatedfor three years without rotation.The area is located in the municipality of Moreira Sales, in the Northwest of Paraná State. The experimental design adopted was randomized blocks with 31 treatments (cotton genotypes and varieties) and 10 replicates. Seeds of each treatment (table 1) were sowed with plant spacing and row spacing of 50 cm. The cultivars IAC-24 and Fiber Max 966 were used as patterns of resistance and susceptibility, respectively, and arranged on the edges of the blocks. The population of M. incognita found in Moreira Sales was also collected and tested in the greenhouse experiments. Data were submitted to variance analysis followed by a grouping analysis based on the Scott Knott methodology at 5%. All the analyses were performed using the Statistic Package Genes (Cruz, 2006).

Quanti cation of J2 in soil samples and physical and chemical analyses of soil
Soil samples were collected for chemical, physical and nematological analyses. Soil components revealed silt, sand and clay contents of 4%, 88% and 8%, respectively. Soil chemical contents were used for fertilizer recommendation. For quanti cation of J2 in soil in the experimentation area, soil samples were collected at 0-20 cm depth before seeding, 60 days after seeding and 120 days after seeding (Figure 1).

Extraction of nematodes
Nematodes were extracted from 100 cc of soil following Jenkins (1964) and from cotton roots according to Coolen and D'Herde (1972). Sieves of 48 and 400 mesh were used for separation of nematodes. Eggs and J2 of M. incognita were quanti ed under a light microscope using a Peters' slide.

Evaluation
For the eld trial the assessment was conducted at 126 days after seeding. Cotton plants were uprooted and the root system rated according to the scale proposed by Khan et al. (2016) (Table 2).

Greenhouse Experiments
The greenhouse experiments revealed that the three populations of M. incognita race 3 were important in the selection of resistant cotton genotypes. In this sense, the population of Umuarama (UMU) was most virulent to 68% of the cotton genotypes, followed by Moreira Sales (MS) to 26% and Iporã (IPO) 6%.
The cultivar IAC 24 behaved as resistant against the populations of MS (mean of RF=0.67) and IPO (mean of RF=0.38) and moderately resistant against the population of UMU (mean of RF=1.75). Therefore, UMU was the only population presenting virulence to that resistant cultivar. The susceptibility of cv. Fiber Max 966 was con rmed by the high reproductive rates of the three populations of M. incognita race 3 (Table 3).
The RF obtained for the 31 tested genotypes considering the evaluation performed at 120 dai for the three isolates, made possible their ranking into different categories based on their reaction to M. incognita. Genotypes CD05-1222, CD05-419, CD05-1087, CD05-1323 and CD05-1170 were classi ed as R (Table 4), while the remaining was classi ed either as MR, or MS or S (Table 3).
The genotypes CD05-419, CD05-1087, CD05-1170, CD05-1222, CD05-1323 and CD05-945 presented RFs below 1.0 against the three populations of M. incognita race 3 and were classi ed as resistant and selected for future work. Those genotypes whose RF from any of the M. incognita populations was higher than 1.0 were discarded.

Field Experiment
The results obtained in the eld showed that the evaluation by rating scale (Table 2) allowed the separation of the genotypes into 4 groups, according to the Scott Knott test at 5% probability (Table 4). According to the evaluation criterion adopted (gall index), the cotton genotypes CD05-419 (GI= 1.90), CD05-1087 (GI= 1.70), CD05-1170 (GI= 1.70) and CD05-945 (GI= 1.60) were classi ed as resistant due to gall indexes less than 2.0. The other genotypes were classi ed as moderately resistant, moderately susceptible and susceptible. The patterns of susceptibility and resistance, Fiber Max 966 and IAC-24, respectively, presented gall indexes 4.40 and 2.20 and were classi ed as susceptible and moderately resistant (Table 4).
Some cotton genotypes reached different status when data from the eld were compared with data from greenhouse. The genotypes CD05-1222 and CD05-1323 were classi ed as resistant in the greenhouse assay (mean of GI= 5 with RF= 0.37) and moderately resistant in the eld (mean of GI= 2.0). In this case, these genotypes were classi ed as moderately resistant. Despite these differences, some genotypes such as CD05-419 and CD05-1170, maintained the same performance in both conditions.

Discussion
This work, which presents results of the screening of cotton genotypes for resistance to M. incognita may help direct management practices, as it may indicate appropriate cotton genotypes for growing in areas infested with the nematode M. incognita.
In screening assays for nematode resistance, one needs to consider the dynamics of the M. incognita population in the soil. This is because a below-threshold level of nematodes will mask the susceptibility of some genotypes. Therefore, the amount of J2 needs to be closely monitored during the period of experimentation. In the eld experiment, the amount of J2 increased over time as shown in Figure 1, reaching levels which ensure that most of the cotton plants were indeed infected.
The ve isolates of M. incognita race 3, representative for the Northwest region of Paraná state, Brazil, were previously tested for virulence on two resistant Brazilian cotton cultivars (CD 201 and IAC 24) and for aggressiveness on the susceptible cultivar FM 966. Based on this test, the UMU isolate was selected for its virulence to the cultivar IAC 24 and CD 201 and the other two isolates, MS and IPO, for their aggressiveness in the susceptible cultivar FM 966 ( Table 2).
The cultivation of resistant plants in infested areas with root-knot nematodes reduces the population level of the nematode contributing to a good crop yield. However, prolonged use of cotton cultivars with the same resistance gene(s) contribute(s) to the increases in populations of virulent nematode (Roberts, 1995). Ogallo et al. (1997), Zhou et al. (2000), Anwar and McKenry (2007) and Silva et al. (2014). Different strategies have been used for assessing resistance to M. incognita in cotton germplasm collections. Some authors reported the use of single M. incognita isolates to assess cotton genotypes (Davis and May, 2003;Carneiro et al., 2005).

Differences in the virulence of M. incognita isolates on resistant cotton genotypes have been demonstrated by
The use of inoculum from virulent populations of M. incognita with increased reproduction on resistant cotton was reported by Ogallo et al. (1997) when testing cotton germplasm for resistance. Nevertheless, this strategy may complicate the selection of cotton genotypes expressing oligogenic resistance (Zhou et al., 2000).
The populations of M. incognita tested in this study were collected in commercial areas where non-resistant cotton varieties had been cultivated. The use of the three M. incognita isolates, previously tested for aggressiveness and virulence, was very valuable in the selection of resistant cotton genotypes. Indeed, differences in the reaction of some cotton genotypes were found when evaluated under greenhouse and eld conditions. The genotypes CD05-1222 and CD05-1323, for example, were classi ed as resistant in the greenhouse based on the RFs below 1.0 whereas in the eld the same genotypes behaved as moderately resistant (GI=2.0). A non-speci ed difference in behavior has already been reported by Ogallo et al. (1997) for one genotype also tested under both these conditions. Field trials allow a better understanding of the parasite-host-environment interaction, being dependent on the concentration and distribution of the inoculum and on climatic variations (humidity and temperature) during the period of the tests. In the case of the genotypes CD05-1222 and CD05-1323, it is more likely to be due to a case of environmental interaction since there was satisfactory inoculum in the eld, as shown in Figure 1, with an increase of M. incognita population during cotton cultivation. Moreover, the MS isolate tested in the greenhouse experiments was collected in the same area where the eld experiment was run and showed good performance when inoculated onto the standard cotton cultivars for resistance and susceptibility (Table 1).

Conclusions
The results obtained in this work allowed the selection of four cotton genotypes (CD05-945, CD05-1170, CD05-1087 and CD05-419), through greenhouse and eld experiments, for which M. incognita presented a low reproductive rate. Additionally, the selection for virulence and aggressiveness in M. incognita isolates, before performing germplasm screening for resistance, was very important and helpful.  Table4.Host status of cotton genotypes to M. incognitaevaluated under greenhouse and eld conditions. Figure 1 Dynamics of M. incognita population in the eld experiment in Moreira Sales/PR through quanti cation of J2 in soil before seeding, 60 and 120 days after seeding.