Interaction effect of Root Lesion Nematodes and Fusarium culmorum Sacc . on the disease complex on some wheat cultivars

Article Info Abstract: The study was conducted to assess a possible interaction between fungal pathogen of Fusarium culmorum Sacc. and Root lesion nematodes at sequentially and simultaneously inoculation. Fusarium culmorum spore suspension effect evaluated on plant disease severity and nematode density on moderate resistant wheat line to F. culmorum (2-49) and susceptible wheat variety (İkizce) under controlled condition. The disease severity was higher on İkizce cv. than 2-49. This indicates that there is a considerable important pathogen resistance. Simultaneous and sequential inoculation of Pratylenchus thornei and F. culmorum reduced the final nematode density and reproduction rate more than inoculation with only nematode on İkizce cv. The simultaneously P. penetrans and F.culmorum inoculation (N+F) affected the positively of final density on İkizce cv. The N+ F treatment affected the positively of P. neglectus density on İkizce cv., whereas negative effect was found in pre or post inoculation of F. culmorum treatments. No synergistic interactions were detected on 2-49 when plants were co-infected by the root lesion nematode and fungus. It was determined that P. thornei had a positive effect on disease severity when it entered the plant simultaneously and before the F. culmorum on İkizce cv.. The disease severity decreased in F. culmorum was applied four weeks after the P. neglectus treatment on 2-49 wheat line and İkizce cv. In the interaction with F. culmorum on İkizce cv., differences were determined between the species of root lesion nematodes. Received: 07.09.2021 Accepted: 30.12.2021 Online Published: 15.03.2022 DOI: 10.29133/yyutbd.992291


Introduction
Wheat is one of the most important food sources with cultivated approximately 20% land area of the world (Braun et al., 2010). Among the world's wheat producers, Turkey has been important level with 16-21 million tonnes of wheat production. However, there are several biotic and abiotic stress factors that seriously affect wheat production. Pratylenchus neglectus (Rench, 1924) Filipjev Schuurmanns &Stekhoven andP. thornei Sher &Allen, 1953 (Tylenchida: Pratylenchidae) cause significant economic yield losses in wheat by reducing quality and quantity in the world (Smiley and Nicol, 2009). It has been determined that P. thornei causes yield loss of 38-85% in Australia, 12-37% in Mexico, 70% in Israel, 20-32% in Turkey, and 50% in the USA (Armstrong et al., 1993;Gözel, 2001;Nicol and Ortiz-Monasterio, 2004;Smiley et al., 2005;Toktay, 2008). In addition, 36-85% yield losses in the spring wheat associated with P. neglectus populations were reported in Oregon, USA (Smiley et al., 2005;Yan et al., 2010). Pratylenchus penetrans have been recorded in wheat-growing fields of Iran (Ghaderi et al., 2010), Morocco (Mokrini et al., 2016), and Turkey (Yüksel, 1974;. Pratylenchus penetrans affects wheat crops yield by 10-19% in Canada (Nicol and Rivoal, 2008). It has been reported that P. thornei and P. neglectus were found in different densities and mixed populations in wheat fields in different regions of Turkey (Yavuzaslanoğlu et al., 2012;Yavuzaslanoğlu et al., 2020;. Fusarium graminearum (Gibberella zeae) and F. culmorum Sacc. are widespread soil-borne fungi in wheat as root and crown rot diseases and decrease the yield in the world and in Turkey (Miedaner et al., 2008;Poole et al., 2012;Köycü and Sukut, 2018;Erginbaş Orakçı et al., 2018). It was determined that yield losses from F. culmorum in Turkey reached 43% in winter wheat and 54% in durum wheat in the Central Anatolian plateau (Bağcı et al., 2001;Hekimhan et al., 2004). Fusarium culmorum is known to produce toxins and enzymes to cause infection, and these toxins play an important role in the pathogenicity (Hestbjerg et al., 2002;Llorens et al., 2006) and important in the nematode-fungus interaction with synergism or antagonism (Back et al., 2002). Fusarium culmorum spore suspension and culture filtrate had negative effects on root-lesion nematodes in different levels of in-vitro studies (Göze Özdemir et al., 2018 and 2021). Similarly, several studies were reported antagonistic relationships between nematodes and fungi (Sankaralingam and McGawley, 1994;El-Borai et al., 2002a and2002b;Poornima et al., 2007).
Organisms occupying the same niche can interact with each other and are not exceptional for plant pathogenic fungi and nematodes. When plant pathogenic fungi and nematodes occur together on the same plant, they can act independently and have an additive effect on damage and yield, or interact with each other in a synergistic or antagonistic causing more and less damage, respectively (Edin et al., 2019;Viketoft et al., 2020). Root lesion nematodes act intracellularly in the root cortex and create a pathway by breaking the cell wall with their stylet (Castillo and Vovlas, 2007). These breaking cells and wounds help many soilborne diseases and pests to enter the plant (Hoseini et al., 2010;Mallaiah et al., 2014). In several earlier studies, it has been reported that nematode penetration increased enzymes secreted by a soil-borne fungal pathogen in roots (Edmund and Mai, 1967;Nord-Meyer and Sikora, 1983). Soil-borne pathogen infection of roots can result in reduced host resistance, which can lead to larger nematode populations (Taheri et al., 1994;Back et al., 2002;Viketoft et al., 2020).
The aim of this study was to investigate the interaction in disease complex between P. penetrans, P. thornei, P. neglectus species, and F. culmorum on moderate resistance to F. culmorum wheat line and susceptible wheat cultivar in controlled conditions.

Materials
Three root-lesion nematode species, Pratylenchus penetrans, P. thornei, P. neglectus, collected from Isparta province, were purified. Morphological-molecular identifications of these root-lesion nematode species were made by Söğüt and Devran (2011). The cultures of these species are preserved under laboratory conditions, and the mass production of carrot cultures was continued at regular intervals. Fusarium culmorum B4 isolate originated from Adana province (Arıcı, 2006) was used in the study. Fusarium culmorum isolate was reisolated periodically to prevent loss of pathogenicity. Wheat variety 2-49 Moderate resistant to F. culmorum wheat line used breeding material of F. culmorum, spring and bread wheat line (Erginbas-Orakci et al., 2016 and were provided from CIMMYT (International Maize and Wheat Improvement Center). Susceptible İkizce wheat cultivar was obtained from the Department of Crop Sciences in ISUBU (Isparta University of Applied Sciences).

Nematode inoculum
Mass cultures of Pratylenchus penetrans, P. thornei, and P. neglectus were reproduced and maintained on carrot disks (Zuckerman et al., 1985). Root lesion nematode with carrots transferred to 12 cm diam petri dishes by cutting into small pieces and added sterilized water, then waited for approximately 6 hours for nematode extraction. Nematodes were extracted by using 38 and 20 μm sieve into a centrifuge tube. Nematode inoculum composed of larvae+adults was counted under the light microscope, and a thousand larvae + adult nematodes were prepared to use in the experiments.

Fusarium culmorum spore suspension
In the first step, 120 g rye grains were weighed in the jar and added pure water, and then waited for 24 hours. In the second stage, these jars were autoclaved at 1.2 atm for sterilization for 20 minutes and taken to room temperature to cool. In the third step, jars were kept for two days by a stretched-film cover. Then F. culmorum was detected previously pure cultured into potato dextrose agar and was inoculated with 10 discs on rye grains per jar. The inoculated jars were incubated for 10 days. Then, these grains cultured with F. culmorum were taken out of the jar by mixing; 1 g sample was placed into 5 ml sterile water and filtered with a micro cloth into sterile tubes. Spore concentration was counted under the light microscope by using a hemocytometer adjusted to 2500 spores g -1 soil density (Hassan et al., 2012).

Interaction between Root lesion nematodes and Fusarium culmorum on 2-49 moderate resistant and susceptible İkizce wheat cultivars
The study was conducted between 2018-2019. Root lesion nematodes Pratylenchus thornei, P. neglectus, and P. penetrans and wheat lines susceptible İkizce and moderate resistant 2-49 were used in all interaction experiments. All experiments were carried out under controlled conditions with 25±2 °C temperature and 60±5% relative humidity and were conducted randomized block design with 5 replicates. Wheat seeds were sown into each pot, including autoclaved ca. 200 g sandy soil. Ten days after wheat germination, wheat germination, root-lesion nematodes, or F. culmorum were applied to ca. 3 cm soil surface depth around the root zone with plastic pipettes. Six treatments were made up simultaneously and sequentially to determine interactions. Treatments were; untreated control (C), only F. culmorum inoculation (F), only root-lesion nematode inoculation (N), simultaneously inoculation of root-lesion nematode and F. culmorum (N+F), First F. culmorum inoculation-sequentially four weeks after root-lesion nematode inoculation (F+4N) and first root-lesion nematode inoculation -sequentially four weeks later F. culmorum inoculation (N+4F). Initial root-lesion nematode density (Pi) was 1000 eggs+larvae+adult each pot -1 in all experiments. Fusarium culmorum, 2500 spores g -1 soil were used in the experiment, then watered with tab-water according to Hasan et al. (2012). Plants were removed from soil approximately 10 weeks later and washed with tab-water. Disease severity of F. culmorum on the root and root coller was evaluated 0-4 disease index reported by Wildermuth and McNamara, (1994) where 0 = completely healthy, 1 = less than 25% necrosis, 2 = 25-50% necrosis, 3 = 50-75% necrosis and 4 = greater than 75% necrosis. Final root-lesion nematode density (Pf) in soil and root was determined by extraction technique of Baermann funnel and counted under the light microscope. Additionally, at the end of the study, nematode reproduction rates [PF (final) / PI (first)] were recorded.

Statistical analysis
Disease severity of F. culmorum, final root-lesion nematode density, the reproduction rate of each root-lesion nematode species, and plant growth parameters were determined by using SPSS 20.0 (Illionis, US.) analysis of variance (ANOVA). Treatments were compared with TUKEY to determine the differences between means at 0.05 significance levels. Also, a t-test was conducted to determine whether the susceptibility of the wheat was important in the evaluation parameters of each application (p≤0.05).

Interaction effect of Pratylenchus neglectus and Fusarium culmorum B4 isolate
In this study, it was found that N+4F treatment had the lowest disease severity with 1.8, and statistically, the difference was determined between F, N+F, and F+4N treatments on 2-49 wheat line (p<0.05). Final nematode density and reproduction rate were close to each other in N, N+F, N+4F, and F+4N treatments on 2-49 wheat lines (p<0.05). The lowest disease severity with 2.6 indexes was observed on N+4F (P<0.05) treatment on susceptible İkizce cv., while F, N+F, and F+4N had the highest disease severity with 4.0 index. The final population density and reproduction rate of P. neglectus in N and N+F treatment had higher than F+4N and N+4F treatments on susceptible İkizce cv. Interestingly, the final nematode density and reproduction rate of N+4F treatment was found extremely low level compared to N+F treatment. In the pre-contamination application of F. culmorum, the population density of P. neglectus decreased compared to the post-contamination application statistically in the Table 1.

5
Letters showed statistical difference among means at 0.05 significance level.
It was determined that whether the wheat is resistant or susceptible to F. culmorum is important in the disease severity parameter in F, N+F, N+4F, and F+4N treatments. However, it was found that wheat's resistance and susceptibility to F. culmorum were important only in N+4F treatment in final nematode density and reproduction rate parameters (Table 2).

Interaction effect of Pratylenchus penetrans and Fusarium culmorum B4 isolate
No statistically significant difference was found among treatments on 2-49 lines in disease severity, final nematode density, and nematode reproduction rate. The disease severity was found 4.0 index in F, N+F, N+4F, and F+4N applications on İkizce cv. No statistically significant difference was found between N, F+4N, and N+4F applications in total nematode density and reproduction rate on İkizce cv (p≥0.05). Total nematode density and reproduction rate were found 3402.4 nematodes root -1 +200 g soil and 3.3, respectively in N+F applications that the highest value among the applications on İkizce cv. It appears that simultaneous nematode and fungus inoculation on İkizce cv affects the P. penetrans density positively (Table 3).  2 0 = completely healthy, 1 = less than 25% necrosis, 2 = 25-50% necrosis, 3 = 50-75% necrosis and 4 = greater than 75% necrosis.  Reproduction rate = final nematode density / initial nematode density.

5
Letters showed statistical difference among means at 0.05 significance level.
It was determined that whether the wheat is resistant or susceptible to F. culmorum was important in the disease severity parameter in F, N+F, N+4F and F+4N treatments. However, it was found that wheat's resistance and susceptibility to F. culmorum was important in N+F and N+4F treatments in final nematode density and reproduction rate parameters (Table 4).

Interaction effect of Pratylenchus thornei and Fusarium culmorum B4 isolate
The statistical difference was found in disease severity in N+F (2.6 indexes) and N+4F (1.6 indexes) treatments on 2-49 lines. It was determined that disease severity decreased in the N+4F treatment on the 2-49 lines. It appears that before inoculation of the P. thornei affects the density of F. culmorum on the 2-49 lines. No statistical difference was found among treatments on 2-49 lines in final nematode density and nematode reproduction rate. While the highest disease severity was found 4.0 and 3.6 indexes in N+4F and N+F treatments on İkizce cv., the lowest was found 2.2 indexes in F+4N. The final nematode density and reproduction rate on İkizce cv. were higher N and N+4F treatments than N+F and F+4N treatments. The lowest reproduction rate was found in F+4N treatment on İkizce cv. It is seen that the density of P. thornei decreases in all applications where fungus and nematodes are together on İkizce cv. (Table 5). It was determined that whether the wheat is resistant or susceptible to F. culmorum is important in the disease severity parameter in F, N+F and N+4F treatments. However, it was found that wheat's resistance and susceptibility to F. culmorum was important only in N and F+4N treatment in final nematode density and reproduction rate parameters (Table 6).

Discussion
It was observed that wheat susceptibility of Fusarium culmorum directly affects disease severity according to the results of the t-test. The disease severity was found to be lower on 2-49 wheat line than İkizce cv. in all experiments. This suggests that the use of pathogen-resistant plants is important to reduce the damage caused by pathogens. However, there were differences in treatments where plant susceptibility of F. culmorum was important in final nematode density and reproduction rate parameters according to the results of the t-test. In the study, the resistance of the plant to the F. culmorum was found to be important in terms of nematode development. The final nematode density and reproduction rate changed in treatments where root lesion nematode and F. culmorum together on İkizce susceptible variety. Pre or post inoculation of F. culmorum can increase or decrease the final nematode density and reproduction rate. The wheat susceptibility of F. culmorum in final nematode density and reproduction rate parameters was found to be statistically significant only in N+4F treatment in P. neglectus and F. culmorum B4 disease complex, whereas it was found significant in N and F+4N treatments in P. thornei and F. culmorum B4 disease complex. Final nematode density and reproduction rate of N+4F treatment in 2-49 wheat line were higher than İkizce cv. in P. neglectus and F. culmorum B4 disease complex. In P. thornei and F. culmorum B4 disease complex, Final nematode density and reproduction rate of N treatment on 2-49 wheat line were lower than İkizce cv. However, The F+4N treatment's final nematode density and reproduction rate on İkizce cv. were lower than 2-49 wheat line. The wheat susceptibility of F. culmorum in final nematode density and reproduction rate parameters were found to be statistically significant in N+F, and N+4F treatments in P. penetrans and F. culmorum B4 disease complex and final nematode density and reproduction rate of N+F treatment on 2-49 wheat line were lower than İkizce cv. However, The N+4F treatment's final nematode density and reproduction rate on İkizce cv were higher than 2-49 wheat lines. As reported by Castillo et al. (2003) the reaction of two different Meloidogyne artiellia populations with partially and very resistant chickpea lines and cultivars were investigated under controlled conditions. They found that M. artiellia infection generally significantly increased the severity of Fusarium wilt in partially resistant genotypes, but in highly resistant lines, it was determined that the resistance could be broken with increasing fungal inoculum density in the presence of nematodes. In the presence of Root lesion nematodes on 2-49 wheat line, no increases in disease severity was determined, and it was found that the resistance of F. culmorum was not broken. Ahmadi et al. (2021) investigated the interactions among Heterodera filipjevi, F. culmorum, and drought on a set of wheat germplasm with differing levels of resistance/tolerance to cereal cyst nematode, crown rot, and drought. They found that co-occurrence of water stress and H. filipjevi increased the cyst density of wheat plants, particularly in accessions susceptible to Heterodera, while co-inoculation of F. culmorum and H. filipjevi reduced cyst density. Also, 2-49 (resistant to F. culmorum) plants were the least affected in all treatments of H. filipjevi and F. culmorum. The simultaneous nematode and fungus inoculation in the disease complex affected the positively of P. neglectus density on İkizce cv., whereas a negative effect was found in pre or post inoculation of F. culmorum treatments. Pratylenchus penetrans density increased nematode and fungus inoculation on İkizce simultaneously. In P. thornei and F. culmorum disease complex, the nematode density and reproduction rate decreased in all treatments where fungus and nematodes were together on İkizce. No interaction was detected between P. thornei, P. penetrans, P. neglectus species, and F. culmorum on 2-49 wheat lines. Root lesion nematodes were found to reproduce on İkizce cv. wheat variety and 2-49 wheat line. However, the development of F. culmorum differentiated on İkizce cv. and 2-49, and these differences affected the interaction between nematodes and fungi. This result shows that the density of F. culmorum is important in the interaction between nematodes and fungi.
In the present study, while there was no change in final nematode density of P. neglectus and P. thornei in F. culmorum was applied 4 weeks after the nematode treatment (N+4F) compared to only nematode inoculation on 2-49 wheat lines, it was observed that disease severity decreased. However, on İkizce cv., the final nematode density of P. neglectus decreased in N+4F treatment compared to only nematode inoculation, and accordingly, disease severity was found decreased. In contrast, the disease severity decreased in P. thornei inoculation 4 weeks after the application of F. culmorum (F+4N), increased in N+4F treatment on İkizce cv. Interestingly, it was determined that P. thornei final nematode density was lower than only P. thornei treatment in N+4F and F+4N treatments. It was determined that P. thornei had a positive effect on disease severity when it entered the plant simultaneously and before the F. culmorum. Hajihassani et al. (2013) investigated the interactions of Heterodera filipjevi and F. culmorum at different inoculation times in the Sardari winter wheat variety in 2009 and 2010 under field conditions, and it was observed that the nematode was suppressed in H. filipjevi application 4 weeks after the F. culmorum application. Hassan et al. (2012) have investigated the interaction of H. avenae and F. culmorum in Sham 3 durum wheat under greenhouse conditions, and as a result of the experiment, it was stated that which organism entered the plant first was important. It was found that P. sudanensis was adversely affected when F. oxysporum inoculated before nematode (Saadabi and Yassin 2007).
Differences have been determined among Root lesion nematode species in nematode-fungus interaction. The different pathogenicity of nematode species, cuticle permeability, and biochemical differences between nematode species may have affected this situation. When only N applications were examined, the reproductive rate of P. penetrans was found to be lower than P. neglectus and P.thornei. While parthenogenesis is observed in most of the Pratylenchus species, sexual reproduction is observed in P. penetrans (Castillo and Vovlas, 2007). Previous studies revealed that there was biodiversity in terms of pathogenicity even among populations of the same species, including P. brachyurus (Payan and Dickson, 1990), P. goodeyi and P. penetrans (Hafez et al., 1999), andP. vulnus (Pinochet et al., 1994) species are reported (Mudiope et al., 2004). Göze Özdemir (2021) reported that two out of five populations of P. thornei had a higher reproduction rate. Furthermore, it has been reported that the cuticle of nematode species affects the penetration of compounds and that biochemical differences between nematode species cause detoxification or degradation of these compounds (Tsoo and Yu, 2000). In the present study, While P. neglectus and P. penetrans were synergistically in relationship with F. culmorum in simultaneous nematode and fungus inoculation, P. thornei was an antagonistic relationship with F. culmorum in all treatments. Riedel et al. (1985) and Bowers et al. (1996) reported that Verticillium dahliae developed in the presence of the P. penetrans population in potatoes; however, it did not disease formation with P. crenatus and P. scribneri presences. Hafez et al. (1999) reported that the P. neglectus population in Canada had a synergistic relationship with V. dahliae, whereas the Parma and Idaho populations did not have a disease complex with V. dahliae and had no yield losses. Bhattarai et al. (2009) found that Rhizoctonia solani damage increased in the combination of Globodera pallida with R. solani or G. rostochiensis with R. solani, and stem canker index increased significantly in coinoculation with G. pallida and R. solani compared with R. solani alone.
The results of this study clearly show that the interaction between Root lesion nematode species and F. culmorum was affected by multiple factors and synergistic or antagonistic interaction changes according to the treatments difference.Unlike previous studies was found disease severity of F. culmorum did not increase in the presence of the Root lesion nematode species in all treatments on İkizce wheat in the study. The density of F. culmorum used in the study may have also been effective in this result. The interaction effect may change with increasing fungal inoculum densities, which constitutes a separate study subject. Gao et al. (2006) found that reproduction of Heterodera glycines was significantly reduced by high levels of F. solani, and severe root necrosis was observed when soybean plants were co-inoculated. In addition, it was determined that F. culmorum was effective on nematode density on İkizce cv. Here, the plant defense mechanism should not be overlooked. The reason why the treatment priority of the organism (nematode or fungus) was important in interaction on İkizce may be that the secretions of the organism entering the plant first act as an effector and stimulate the plant defense mechanism. Also, the fact that no interaction could be determined between F. culmorum and Root lesion nematodes species on 2-49 wheat line once again showed the importance of plant resistance in control.
In this study, it was shown that both organisms cause significant yield loss in cultivated cereal areas. It is important to consider both pathogens when designing control methods. The priority is to prevent the contamination of the field and the spread of the two organisms. It is seen that it is important to suppress both with early applications in disease complexes. Therefore, are recommended different methods should be established to suppress the population of both pathogens in the field.