Interactions among cereal cyst nematode Heterodera filipjevi, dryland crown rot Fusarium culmorum, and drought on grain yield components and disease severity in bread wheat

Abstract

The cereal cyst nematode (CCN) Heterodera and crown rot caused by Fusarium species limit cereal production and yield potential. Losses increase when CCN and crown rot occur together, especially under water stress conditions. The objective of this study was to investigate the interactions among Heterodera filipjevi, Fusarium culmorum, and drought on a set of wheat germplasm with differing levels of resistance/tolerance to CCN, crown rot and drought. Plant emergence was significantly reduced (56% seedling death) when seeds were planted in a water stress environment where F. culmorum and H. filipjevi were present. Crown rot was more severe under water stress compared with Fusarium inoculation alone. The number of cysts fell significantly when H. filipjevi was co-inoculated with F. culmorum, revealing an antagonistic interaction between the nematode and fungus. The highest number of cysts was found on the susceptible accession ‘Seri’ under water stress conditions. Water stress caused a significant reduction in plant height, while F. culmorum inoculation did not, unless combined with water stress. Yield components were significantly reduced by each of the single stresses and losses were greater when plants were exposed to double or triple stresses. Water stress and F. culmorum inoculation caused a significant reduction in spike weight and seeds per spike. The chlorophyll content of wheat leaves was negatively affected by water stress and inoculation by both nematode and fungus. In conclusion, water stress exacerbates the damage caused by CCN and crown rot, and planting drought-resistant varieties would be an ideal solution to reduce losses.

Résumé

Le nématode à kyste des céréales (NKC) Heterodera et la pourriture du collet causée par des espèces de Fusarium limitent la production des céréales et le potentiel de rendement. Les pertes s’accroissent lorsque le NKC et la pourriture du collet agissent simultanément, particulièrement dans des conditions de stress hydrique. Le but de cette étude était d’explorer les interactions entre Heterodera filipjevi, Fusarium culmorum et la sécheresse sur une série de germoplasmes de blé affichant divers niveaux de résistance ou de tolérance au NKC, à la pourriture du collet et à la sécheresse. L’émergence des plants était significativement réduite (taux de mortalité de 56% des plantules) quand les graines étaient semées dans un environnement sujet aux stress hydriques déjà colonisé par F. culmorum et H. filipjevi. La pourriture du collet était plus grave dans des conditions de stress hydrique que lorsqu’elle était causée par Fusarium uniquement. Le nombre de kystes chutait radicalement lorsque H. filipjevi était inoculé à la fois avec F. culmorum, révélant une interaction antagoniste entre le nématode et le champignon. Le plus grand nombre de kystes a été trouvé chez l’accession réceptive ‘Seri’ dans des conditions de stress hydrique. Ce dernier a réduit substantiellement la taille des plants, tandis que l’inoculation avec F. culmorum n’a eu aucun effet à moins d’être combinée au stress hydrique. Les composantes du rendement étaient significativement réduites par chacun des stress individuels et les pertes étaient plus grandes lorsque les plants étaient exposés à deux ou trois stress. Le stress hydrique et l’inoculation avec F. culmorum ont réduit significativement le poids de l’épi et le nombre de graines par épi. La teneur en chlorophylle des feuilles des plants de blé a été influencée négativement par le stress hydrique ainsi que par les inoculations de nématodes et de champignons. Finalement, le stress hydrique exacerbe le dommage causé par le NKC et la pourriture de collet et, en conséquence, une solution idéale pour réduire les pertes serait de semer des variétés résistantes à la sécheresse.

Keywords:

• cereal cyst nematode
• Fusarium
• Heterodera
• interactions
• soilborne pathogens
• water stress

Mots clés:

• Nématode à kyste des céréales
• Fusarium
• Heterodera
• interactions
• agents pathogènes terricoles
• stress hydrique

Introduction

Wheat (Triticum aestivum L.), one of the world’s staple food crops, is grown globally across most cropping regions due to its adaptability to different climates (Dababat et al. 2014a). However, commercial crop production is highly dependent on growing conditions. With average temperatures increasing globally, the availability of soil water across different regions of the world is declining (Compant et al. 2010). Recent evidence shows that global warming negatively impacts soil organisms and ecosystems and increases the chance of expanding the virulence of a number of plant pathogens (Garrett et al. 2006). Research also shows that the co-occurrence of biotic and abiotic stresses as limiting factors to grain production is inevitable (Compant et al. 2010; Ramegowda and Senthil-Kumar 2015). Plant reactions to biotic and abiotic stresses are complex and consist of a great number of molecular, physiological and cellular responses (Foyer and Noctor 2005; Swindell 2006; Ramirez et al. 2009; Šamajová et al. 2013; Rejeb et al. 2014). Recent studies have suggested that a combination of biotic and abiotic stresses disrupts plant physiological and metabolic reactions, resulting in losses in fitness and productivity (Anderson et al. 2004; Yasuda et al. 2008; Atkinson and Urwin 2012; Rejeb et al. 2014).

All biotic and abiotic stresses are limiting factors to agricultural production. The combination of drought with heat stress is a major limiting factor in arid and semi-arid wheat-growing areas (Rizhsky 2004). Ingram and Bartels (1996) forecasted that the lives of approximately 1.8 billion people would be endangered due to drought or water limitations by the year 2025.

Cereal cyst nematodes (CCNs) of the genus Heterodera (Dababat and Fourie 2018b) and Fusarium spp. causing dryland root rot (Nicol et al. 2010) are major soilborne pathogens (SBPs) causing significant yield losses to wheat in dryland regions. Potential losses are more pronounced when diseased plants are under water stress conditions (Dababat et al. 2018a). Co-occurrence of other biotic and abiotic stresses with CCNs accentuate significant economic yield losses (Dababat et al. 2014b).

Heterodera filipjevi is one of the most economically important CCN species reported to infect crops in multiple regions of the world (Dababat et al. 2014b; Seid et al. 2021). There are some reports on the predominance of H. filipjevi, compared with other CCN species, in most cereal growing fields of Iran (Damadzade and Ansaripour 2001; Tanha Maafi et al. 2007; Seifi and Karegar Bide 2013). Fusarium culmorum is a SBP that causes root rot and crown rot in cereals, particularly in wheat and barley (Scherm et al. 2013; Erginbas-Orakci et al. 2016; Orakci et al. 2018). Humid conditions accelerate the initial development of the disease at the seedling stage, while water stress significantly accelerates yield loss and disease severity in late stages (Chekali et al. 2011).

Since CCNs and root rot fungi occupy the same rhizosphere, especially under cereal monoculture farming, a synergistic interaction may cause greater yield loss and root damage than the occurrence of each pathogen alone (Hassan et al. 2012). In addition, interaction between plant parasitic nematodes and fungi can increase disease severity as a result of the disease complex they can create (Nicol et al. 2010; Dababat et al. 2018a). Plants attacked by nematodes may modify their physiology and change their basal defences, resulting in increased susceptibility to other pathogens (Riedel 1988). Hajihassani et al. (2013) observed that simultaneous infection of plants by both H. filipjevi and F. culmorum had a greater effect on morphological parameters and caused 50% reduction in wheat grain yield, while individual infection caused reductions of only 26% and 36%, respectively. The severity of F. culmorum increased when co-inoculated with H. filipjevi, while F. culmorum treatment reduced the H. filipjevi population (Hajihassani et al. 2013).

Gao et al. (2006) investigated the interaction between two soybean SBPs, Fusarium solani f. sp. glycines and the soybean cyst nematode Heterodera glycines. They found that reproduction of H. glycines was significantly reduced by high levels of F. solani and severe root necrosis was observed when soybean plants were co-inoculated with high levels of both SBPs. Bhattarai et al. (2009) studied the interaction between three potato diseases: Globodera pallida, Globodera rostochiensis (cyst nematodes) and the soilborne fungus Rhizoctonia solani, and found that the combination of G. pallida with R. solani or G. rostochiensis with R. solani resulted in greater damage by R. solani and that the stem canker index increased significantly under co-inoculation with G. pallida and R. solani compared with R. solani alone.

Mayek-Perez et al. (2002) studied the interaction between drought and Macrophomina phaseolina (a causal agent of charcoal rot) in the common bean and observed that drought stress accelerated charcoal rot development and increased disease severity. In addition, M. phaseolina increased the negative effect of drought stress on foliar relative water content. A similar study conducted by McElrone et al. (2001), on the effect of water stress on infection by Xylella fastidiosa (a casual agent of leaf scorch) of Parthenocissus quinquefolia plants, found that symptoms of bacterial leaf scorch were more severe under low water than high water conditions. Similarly, spread of Chalara paradoxa was amplified by drought stress in the date palm (Suleman et al. 2001).

Jenkins and Coursen (1957) studied the effect of the root-knot nematodes Meloidogyne incognita acrita and M. hapla on Fusarium wilt of tomato. They found that inoculation of plants with both nematodes increased wilt. Co-infection of M. incognita and Fusarium resulted in 100% wilting of the tomato plants, compared with 60% when M. hapla was inoculated alone. This means that greater damage is caused by the nematode-fungus complex, compared with the cumulative effects of the individual pathogens.

In contrast, an antagonistic interaction can occur between fungi and nematodes where the fungus reduces the hatching of nematode cysts and eggs, thereby reducing invasion of plants by the juveniles (Powell 1971; Duncan 1991). Some fungi can infect the nematode eggs. For example, Verticillium chlamydosporium infects the eggs of H. avenae only two to three days after cyst hatching (Ragozzino and D’Errico 2011), while F. culmorum has been shown to reduce the H. avenae cyst population (Hassan et al. 2012).

The co-occurrence of soilborne pathogens and drought can lead to a negative effect on plants. Soilborne pathogens such as nematodes affect water relations in plants by decreasing water use efficiency and water consumption (Audebert et al. 2000). On the other hand, drought stress can reduce nematode movement and population in the soil (Davis et al. 2014).

Plants that were exposed to drought stress during early stages of development showed an increase in abscisic acid (ABA) and reactive oxygen species (ROS) production, resulting in a suppression of pathogen infection (Fujita et al. 2006). Early exposure of plants to pathogens may also change plant tolerance to drought stress. When pathogen infection weakens a plants’ basic defences, susceptibility to drought will occur; in contrast, when pathogen infection induces an initial tolerance mechanism, plants will be more drought tolerant (Van Hulten et al. 2006). The objective of this study was to determine the effect of H. filipjevi and F. culmorum under water stress conditions on wheat varieties with differing levels of resistance/tolerance to both pathogens.

Material and methods

The research was carried out by the SBPs Programme at the CIMMYT-Turkey facility located at the Transitional Zone Agriculture Research Institute (TZARI) Eskisehir, Turkey.

Soil, seed disinfestation and germination

A standard potting mixture of sand, field soil, and organic matter was used in the experiments. The sand and the field soil were sieved and autoclaved at 110°C for 2 h, and the organic matter twice at 70°C for 5 h over the course of two successive days. Ten wheat lines including three control accessions, 2–49 (resistant to F. culmorum), ‘Seri’ (susceptible to both pathogens), ‘Silverstar’ (resistant to H. filipjevi), and seven Iranian landraces were selected based on their reactions to the factors being examined (Table 1). Seeds were surface-sterilized with 1% NaOCl for 3 min followed by rinsing three times with deionized water. Surface-disinfested seeds were germinated on moist sterile blotting paper in Petri dishes for 3–4 days at 23°C.

Table 1. List of wheat accessions used in this study and their host status to Heterodera filipjevi, Fusarium culmorum and water stress

Accession	Cross name	CID	GID	Resistant Reaction			Type
				H. filipjevi	F. culmorum	Water stress
P52	IWA8612991	350084	375540	MR-MS*	MR-MS*	MS-S*	Iranian landrace
P116	IWA8613637	268776	284176	S*	S*	S*	Iranian landrace
P159	IWA8613614	350351	375807	S*	S*	S*	Iranian landrace
P279	IWA8613070	350138	375594	MS-S*	S*	S*	Iranian landrace
P396	IWA8609679	349263	374719	MR-MS*	MR-MS*	MS-S*	Iranian landrace
P399	IWA8613163	350207	375663	S*	MR-MS*	MS*	Iranian landrace
P426	IWA8607380	348735	374191	MS-S*	MR*	S*	Iranian landrace
	2–49			S*	R	R*	Bread Wheat
	Seri			S	S	MR*	Bread Wheat
	Silverstar			R	S-HS*	S*	Bread Wheat

Abbreviations stand for: R = Resistant; S = Susceptible; MR: Moderately resistant; MS: Moderately susceptible; HS: Highly susceptible; CID = Cross identification; GID = Germaplam identification.

*Results are from this study.

Greenhouse experimental design and treatments

Pots (14 cm in diameter × 15 cm in length) were filled with 3 kg of potting mixture consisting of sterilized sand, field soil, and organic matter (70:29:1, v/v/v). Four germinated seeds were planted in each pot. Seedlings were treated with experimental treatments (Table 2). Each treatment (Table 2) was replicated three times and the experiments were arranged in a completely randomized design (CRD). Experiments were repeated twice for data validation (in two independent runs). At the tillering stage, each pot received 0.25 g of ammonium nitrate fertilizer. Table 3 shows a list of the different studied parameters and their assessment methodology.

Table 2. List of the treatments and their descriptions used in the study

Treatment	Code	Description
T1	WS	Water stress	Irrigation level was kept at 60% of field capacity (FC) from sowing time until harvest time
T2	WSH	Water stress + H. filipjevi	Irrigation at 60% of FC
T3	WSF	Water stress + F. culmorum	Irrigation at 60% of FC
T4	WSHF	Water stress + H. filipjevi + F. culmorum	Irrigation at 60% of FC + H. filipjevi + F. culmorum
T5	C	Normal irrigation (Control)	Irrigation at 100% of FC
T6	H	H. filipjevi	Inoculation with 400 second stage juveniles (J2) of H. filipjevi
T7	F	F. culmorum	Inoculation with 1 × 10^6 spores of F. culmorum
T8	HF	H. filipjevi + F. culmorum	Inoculation with 400 second stage juveniles (J2) of H. filipjevi and 1 × 10^6 spores of F. culmorum

Table 3. List of the different studied parameters and their assessment methodology

	Parameter	Description
1	F. culmorum crown rot symptoms	Plants grown under greenhouse and growth room conditions were evaluated for seedling resistance (Zadoks growth stage 14). Plants grown under greenhouse conditions were evaluated at maturity and tested for adult plant resistance. Plants were scored for the typical symptoms of browning percentage on the crown (by observing the disease on the crown of the plants) and the main stem (by measuring the disease symptoms on the stem) using numeric scales (1–5): resistant (1: 1–9%), moderately resistant (2: 10–29%), moderately susceptible (3: 30–69%), susceptible (4: 70–89%), and highly susceptible (5: 90–99%) modified from Wildermuth and McNamara (1994) according to Erginbas-Orakci et al. (2016).
2	H. filipjevi cyst number	Number of cysts on roots was assessed by extracting cysts from each pot as per Dababat et al. (2014b).
3	Plant height (cm)	The main tiller from each plant was measured from soil surface to the spike’s top
4	Final plants number (seedling emergence)	Living plants were counted per each pot
5	Seeds per spike	The average number of seeds per spikes from each pot was calculated
6	Thousand kernel weight (TKW)	The average weight of 1000 kernels per each pot was measured
7	Spike weight (g)	The average weight of spikes from each pot was measured
	Total grain yield/pot (g)
8	Measuring chlorophyll content	Chlorophyll content of flag leaf of each plant main tiller was measured with SPAD (model KONICA MINOLTA) at grain filling stage
9	Relative water content (RWC)	The RWC was measured following the method of Pask et al. (2012). One fully expanded leaf from each plant was marked and detached. The top and bottom of all leaves and any dead tissue was also removed, to leave a five cm mid-section, which was immediately placed into the pre-weighed tubes and sealed to avoid any moisture loss. The tubes were then placed in a cooled, insulated container at 10–15^°C. The tubes were transferred to laboratory where all samples were weighed (tube W + FW). Then, one cm of distilled water was added to each tube. The samples were placed in a refrigerator at 4^°C for 24 h (to allow leaves to reach full turgor). The leaf samples were taken out of the tube, and quickly and carefully blot dried with paper towels. Each leaf sample was weighed to ascertain turgid weight (TW), placed in a labelled envelope and dried at 70^°C for 24 h, or until constant mass. Finally, leaf samples were reweighed to ascertain dry weight (DW) to measure relative water content (RWC) using the following equation:$\mathrm{F}\mathrm{W}=\mathrm{t}\mathrm{u}\mathrm{b}\mathrm{e}\mathrm{W}+\mathrm{F}\mathrm{W}-\mathrm{t}\mathrm{u}\mathrm{b}\mathrm{e}\mathrm{W}$$RWC\%=\hspace{0.17em}\left(FW-DW\right)/\left(TW-DW\right)\times 100$ Where FW is leaf fresh weight, DW is leaf dry weight, and TW is leaf turgid weight
10	Rate of seedling death post germination	$\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e}=Deadplants/\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}$

The level of soil saturation (field capacity) was calculated by adding a measured volume of water to the pots until water ran out from the bottom of the pot. Then, the soil was allowed to drain for 24 h before the gravimetric water content was measured. Soil at this level was considered to be at 100% water capacity. If the water level was more than 10% below the required level, the pots were weighed and water was added to bring them to the required capacity. While under water stress, water level was kept at 60% of field capacity from sowing time until maturity time.

Fusarium culmorum inoculum preparation

A monosporic and molecularly identified and validated isolate of F. culmorum from the CIMMYT-SBP collection was grown on synthetic nutrient agar (SNA) for 10 days under fluorescent lights set at a 12-h photoperiod and a temperature of 24 ± 1°C. Polypropylene bags (20 × 48 cm) with aeration filters (‘Unicorn’ brand Amsterdam, the Netherlands) were filled with moist wheat bran and autoclaved at 121°C for 20 min and 103 kPa pressure three times over three consecutive days. After cooling, each bag was inoculated with one plate of a 10-day-old F. culmorum isolate by scraping the spores from the culture surface and suspending them in sterile distilled water (SDW) as per Erginbas-Orakci et al. (2016). The spore suspension was added to the bags containing wheat bran, mixed thoroughly, and incubated for two weeks at 23°C with daily agitation. Wheat bran inoculated with F. culmorum was suspended in distilled water and filtered through two layers of cheese cloth. The spore concentration was adjusted to 1 × 10⁶ spore mL⁻¹, and methyl cellulose (0.1%, v/v) was added to the spore suspension prior to use.

Heterodera filipjevi inoculum preparation

Soil samples were collected from Kirsehir-Yerkoy, Turkey (Latitude 39°37´0´´ Longitude 34°28´0´´), and cysts were extracted using the Fenwick-Can technique (Fenwick 1940). The cysts were collected and surface-sterilized with 0.5% NaOCl for 10 min and rinsed several times in distilled water. The cysts were kept in a refrigerator at 4°C before being transferred to room temperature to enhance hatching (Dababat et al. 2014b). The freshly hatched J2 of H. filipjevi were then used as inoculum in the experiments.

Growth room trials (Assessment of germplasm to F. culmorum and H. filipjevi with disease ratings)

This experiment was conducted in a growth room to assess germplasm for resistance/susceptibility to F. culmorum and H. filipjevi without any interactions. A single germinated seed was transplanted to experimental plastic tubes (Stuewe and Sons, Corvallis, OR, USA) measuring 2.5 cm in diameter × 16 cm in length and filled with a potting mixture of sterilized sand, field soil, and organic matter (50:40:10, v/v/v). One week after sowing, the stem base of each seedling (0.5–1 cm above the soil level, including the coleoptile) was inoculated with F. culmorum spores suspended in water (1 × 10⁶ spores mL⁻¹) (Erginbas-Orakci et al. 2016). Tubes inoculated with the fungus were covered with plastic sheeting for 48 h to maintain high relative humidity.

Similarly, the H. filipjevi test was performed using the plastic tubes as above filled with a potting mixture composed of sterilized sand, field soil, and organic matter (70:29:1, v/v/v). A single germinated seed was transplanted per plastic tube. Two days after transplanting, three holes were made around the stem base of each seedling and inoculated with 400 freshly hatched J2 of H. filipjevi (Dababat et al. 2014b). To increase the efficiency of nematode penetration, plants were irrigated gently during the first week following nematode inoculation.

Plants were grown in a growth room for 42 days (early tillering, Zadoks growth stage 14; Zadoks et al. 1974) with a 16-h artificial photoperiod and maintained at a temperature of 22 ± 3^°C with 70% of relative humidity. Experimental units were arranged in a CRD in five replicates and the experiment was repeated.

The plants inoculated with H. filipjevi were harvested nine weeks after inoculation. Soil from each tube was collected in a 2 L pot filled with water for cyst extraction, while roots were washed on nested sieves with 850 μm and 250 μm mesh sizes to free cysts from the root system following Dababat et al. (2014b). Cysts from both root and soil extractions were collected on the 250-μm sieve and counted under a stereomicroscope.

Experimental design and data analysis

The experiment was conducted and analysed as a 2 × 2 × 2 × 10 factorial with three replicates in a completely randomized design (CRD), repeated in two runs. Treatment combinations of two drought levels (40% stress and non-stress), two nematode levels (inoculation and non-inoculation), two Fusarium levels (inoculation and no inoculation), ten accession levels (1, 2, 3, …, 10) with three replicates of each treatment combination was used. Analysis of variance (ANOVA) and mean comparisons using the least significant difference (LSD) and Duncan methods were performed with SAS (SAS Institute, Cary, NC, USA) Normality was tested with a one-sample Kolmogorov-Smirnov test, and all parameters except ‘Final plant number’, ‘Seed/spike’ and Leaf relative water content (‘RWC’) showed a normal distribution. Crown rot data were ordinal and analysed with the non-parametric Kruskal-Wallis test.

Results

Water stress (WS) significantly affected all the measured parameters except RWC, while H. filipjevi affected all the traits except spike weight, height and RWC (Table 4). Fusarium culmorum affected all parameters except height and RWC. The water stress × accession interaction was significant for all variables. The Heterodera × accession interaction was significant only for spike weight, seeds per spike, total grain yield and chlorophyll content, while the Fusarium×accession interaction was significant for all variables except seeds per spike, height and RWC.

Table 4. Analysis of variance showing the main effects of accessions and individual stressors (Fusarium, Heterodera, water stress) alone and in combination, on various plant and disease parameters

		P value
S.O.V	Df	Final plant NO/Pot	Spike weight	Seed/spike	TKW	Total grain yield/pot	Height	Leaf chlorophyll content	RWC	Cyst density of Heterodera	Crown rot by Fusarium
Water stress	1	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	0.08	-	-
Nematode	1	0.0015	0.88	0.0003	0.06	<.0001	0.12	<.0001	0.76	-	-
Fusarium	1	<.0001	0.004	0.0039	<.0001	<.0001	0.51	<.0001	0.84	-	-
Accession	9	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	-	-
Water stress × Nematode	1	<.0001	0.09	0.0037	0.027	0.088	0.50	0.89	0.01	<.0001	-
Water stress × Fusarium	1	<.0001	0.59	0.73	0.0004	0.054	0.017	0.001	0.44	-	<.0001
Waterstress  × Accession	9	0.029	<.0001	0.017	0.024	0.056	<.0001	<.0001	0.0002	-	-
Nematode × Fusarium	1	0.0002	0.92	0.74	0.25	0.28	0.34	0.18	0.031	0.034	0.52
Nematode × Accession	9	0.6256	0.05	0.02	0.25	0.002	0.53	<.0001	0.36	0.0004	-
Fusarium×Accession	9	<.0001	0.002	0.62	0.073	0.011	0.57	<.0001	0.51	-	<.0001
Water stress × Nematode × Fusarium	1	<.0001	0.80	0.32	0.24	0.017	0.31	<.0001	0.34	0.092	0.91
Water stress × Nematode × Accession	9	0.2233	0.11	0.59	0.15	0.79	0.47	0.19	0.25	0.29	-
Water stress × Fusarium × Accession	9	0.42	0.0006	0.99	0.05	<.0001	0.66	0.20	0.44	-	0.71
Nematode × Fusarium × Accession	9	0.57	0.09	0.003	0.43	0.01	0.29	0.10	0.31	0.08	0.82
Water stress × Nematode × Fusarium ×Accession	9	0.52	0.76	0.58	0.97	0.27	0.99	0.73	0.42	0.26	0.33
CV %		15.01	15.04	15.03	9.53	17.82	8.30	6.62	21.76	5.85	21.35

Effect of water stress (WS) on accessions compared to the control treatment

There was a significant interaction between water stress and accessions for all parameters measured, indicating that not all accessions responded equally to water stress (Table 4). The effects of WS on trait expression are shown in Table 5. Spike weight was significantly reduced for P159 and P279 by 34% and 32%, respectively, compared with the control treatment, while 2–49 (resistant to F. culmorum), ‘Silverstar’ (resistant to H. filipjevi) and P396 showed no significant reduction. Seeds per spike was significantly reduced compared with the control treatment in half of the accessions, especially in P159 and P279, which were reduced by 37% and 40%, respectively. A reduction in total grain yield per pot was observed for half of the accessions. Plant height was significantly reduced for P52, P279, P426 and 2–49 compared with the control. Water stress caused a significant reduction in leaf chlorophyll content for most accessions except 2–49 and P399. All accessions, except P52, P116, P279, 2–49 and ‘Silverstar’, showed a significant reduction for RWC. Overall, P159 showed the largest change in growth parameters and grain yield components under water stress, while 2–49 and ‘Silverstar’ showed the smallest changes.

Table 5. Effect of water stress (WS) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the stressed treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.96ns	0.98ns	0.92ns	0.88ns	0.83ns	0.96ns	0.99ns	0.99ns	0.96ns	0.99ns
Spike weight	0.75*	0.76*	0.66*	0.68*	0.84ns	0.69*	0.71*	0.92ns	0.86*	0.87ns
Seed/spike	0.73*	0.77*	0.63*	0.60*	0.67*	0.76ns	0.79ns	0.86ns	0.88ns	0.83ns
Thousand kernel weight	0.94ns	0.99ns	0.97ns	0.92ns	0.98ns	0.99ns	0.92ns	0.99ns	0.94ns	0.85ns
Total grain yield/pot	0.62*	0.69*	0.63*	0.77ns	0.79ns	0.65*	0.74ns	0.72*	0.89ns	0.92ns
Height	0.82*	0.93ns	0.90ns	0.84*	0.89ns	0.89ns	0.83*	0.87*	0.99ns	0.98ns
Leaf chlorophyll content	0.90*	0.84*	0.86*	0.82*	0.77*	0.93ns	0.66*	0.95ns	0.64*	0.69*
Relative water content	0.99ns	1.00ns	0.82*	1.00ns	0.77*	0.84*	0.74*	0.96ns	0.77*	1.00ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of F. culmorum stress on accessions

There were significant interactions between Fusarium and accessions for final plants/pot, spike weight, TKW, grain yield and leaf chlorophyll content (Table 4), indicating that not all accessions respond equally to Fusarium stress. As shown in Table 6, Fusarium stress reduced the final plants/pot for P116, P159, P279 and P399 by 35%, 37%, 25% and 50%, respectively. Fusarium stress reduced spike weight by 31% in P399 and 17% in ‘Seri’, an accession susceptible to Fusarium. Seeds per spike were significantly reduced only in ‘Seri’ by 14%. Fusarium stress significantly reduced TKW in P159 and P399 by 15% and 14%, respectively. Fusarium stress only significantly reduced total grain yield per pot for P399, with a 51% reduction. Fusarium stress decreased leaf chlorophyll content in most accessions. Overall, P159 and P399 were the most susceptible to Fusarium stress, while 2–49 and P426 were the most resistant.

Table 6. Effect of inoculation with Fusarium culmorum (F) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the inoculated treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.82ns	0.65*	0.63*	0.75*	0.88ns	0.50*	0.91ns	0.96ns	0.92ns	0.96ns
Spike weight	0.89ns	0.99ns	0.99ns	0.97ns	0.90ns	0.69*	0.90ns	0.85ns	0.83*	0.99ns
Seed/spike	0.93ns	0.91ns	0.98ns	0.89ns	0.86ns	0.90ns	0.88ns	0.97ns	0.86*	0.88ns
Thousand kernel weight	0.90ns	0.87ns	0.85*	0.92ns	0.99ns	0.86*	0.99ns	0.96ns	0.90ns	0.91ns
Total grain yield/pot	0.83ns	0.90ns	0.85ns	0.90ns	0.99ns	0.49*	0.94ns	0.97ns	0.92ns	0.98ns
Height	0.98ns	0.99ns	0.99ns	0.97ns	0.99ns	0.94ns	0.98ns	0.97ns	0.99ns	0.97ns
Leaf chlorophyll content	0.95ns	0.95ns	0.76*	0.89*	0.85*	0.89*	0.94ns	0.97ns	0.70*	0.80*
Relative water content	0.94ns	0.95ns	0.89ns	0.93ns	0.90ns	0.99ns	0.88ns	0.90ns	0.95ns	0.89ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of H. filipjevi stress on accessions

There were significant interactions beween Heterodera and accessions for spike weight, seeds per spike, total grain yield and leaf chlorophyll content (Table 4), indicating that not all accessions responded equally to Heterodera stress, especially for the grain parameters. As shown in Table 7, Heterodera stress reduced seeds per spike by 13% for ‘Seri’, an accession susceptible to H. filipjevi. Among all accessions, only P116 showed a significant reduction in total grain yield with a 40% reduction. Heterodera stress decreased leaf chlorophyll content for most accessions except for P279, P399 and 2–49. Among all accessions, P426, 2–49 and ‘Seri’ showed a reduction for RWC under Heterodera stress. Overall, P116 and Seri were more susceptible to Heterodera stress.

Table 7. Effect of inoculation with Heterodera filipjevi (H) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the inoculated treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.99ns	0.91ns	0.99ns	0.99ns	0.99ns	0.99ns	0.99ns	0.98ns	0.98ns	0.99ns
Spike weight	0.83ns	0.84ns	0.91ns	0.91ns	0.99ns	0.99ns	0.99ns	0.99ns	0.91ns	0.99ns
Seed/spike	0.92ns	0.84ns	0.86ns	0.80ns	0.88ns	0.87ns	0.91ns	0.93ns	0.87*	0.92ns
Thousand kernel weight	0.92ns	0.89ns	0.99ns	0.99ns	0.98ns	0.99ns	0.99ns	0.98ns	0.99ns	0.98ns
Total grain yield/pot	0.79ns	0.60*	0.91ns	0.89ns	0.85ns	0.91ns	0.92ns	0.90ns	0.96ns	0.95ns
Height	0.90ns	0.99ns	0.98ns	0.94ns	0.99ns	0.98ns	0.98ns	0.94ns	0.99ns	0.91ns
Leaf chlorophyll content	0.88*	0.91*	0.76*	0.92ns	0.85*	0.98ns	0.91*	0.94ns	0.78*	0.85*
Relative water content	0.94ns	0.97ns	0.99ns	0.90ns	0.90ns	0.95ns	0.84*	0.78*	0.78*	0.90ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of combined water stress and H. filipjevi (WSH) stress on accessions

When looking at the three-way interaction between water stress, Heterodera stress and accessions, there were no significant interactions for all parameters measured, indicating that all accessions responded similarly to the combined stress (Table 4). As shown in Table 8, WSH stress reduced spike weight and seeds per spike for P279 by 37% and 42%, respectively. Total grain yield per pot also significantly decreased under WSH. The highest reduction in total grain yield per pot was observed for P116 and P399, at 50% and 54%, respectively. WSH reduced the height of most accessions except for P116, P396, ‘Seri’ and an accession resistant to Heterodera (‘Silverstar’), which did not show any significant reduction in plant height in response to water stress or by Heterodera stress treatment. RWC decreased in P52, P396, P399, P426 and ‘Seri’. The accessions P426 and Seri had the highest reduction in leaf chlorophyll content (LCC), 38% and 40%, respectively. Generally, dual WSH treatment caused a reduction in all parameters greater than that caused by WS or H alone. Overall, P159, P279 and P399 were the most susceptible and showed the highest reductions in growth and yield parameters under WSH treatment, while 2–49 was the most resistant.

Table 8. Effect of water stress and inoculation with Heterodera filipjevi (WSH) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the stress treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.99 ns	0.99 ns	0.96 ns	0.92 ns	0.88 ns	0.88 ns	0.99 ns	0.99 ns	0.99 ns	0.99 ns
Spike weight	0.84 ns	0.74*	0.67*	0.63*	0.90 ns	0.64*	0.79 ns	0.99 ns	0.90 ns	0.98 ns
Seed/spike	0.69*	0.81*	0.67*	0.58*	0.73*	0.67*	0.74*	0.80 ns	0.91 ns	0.83 ns
Thousand kernel weight	0.91ns	0.91ns	0.89ns	0.95ns	0.98ns	0.99ns	0.99ns	0.99ns	0.97 ns	0.87 ns
Total grain yield/pot	0.81ns	0.50*	0.70*	0.70*	0.84ns	0.46*	0.79*	0.94ns	0.91ns	0.93ns
Height	0.86*	0.88ns	0.88*	0.88*	0.94ns	0.87*	0.87*	0.84*	0.99ns	0.93ns
Leaf chlorophyll content	0.75*	0.82*	0.67*	0.73*	0.76*	0.93ns	0.62*	0.95ns	0.60*	0.68*
Relative water content	0.86*	0.99ns	0.89ns	0.99ns	0.73*	0.79*	0.73*	0.90ns	0.74*	0.99ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of water stress combined with F. culmorum (WSF) stress

When looking at the three-way interaction between Fusarium, water stress and accessions, there was a significant interaction for spike weight, TKW and total grain yield, indicating that Fusarium and water stress interacted for grain and yield paramters (Table 4). As shown in Table 9, the final plant number was significantly reduced by WSF for all accessions except for 2–49 (resistant to Fusarium), ‘Silverstar’ (resistant to Heterodera), and ‘Seri’ (susceptible to both Heterodera and Fusarium). P396 and P399 showed the highest reduction in plant number, at 50% and 54%, respectively, compared with the control. WSF significantly reduced spike weight and seeds per spike. P279 showed the greatest reduction in spike weight and seeds per spike (49% and 50%, respectively), while 2–49 did not show any significant reduction in spike weight and seeds per spike under WSF treatment. Dual inoculation of WSF reduced TKW significantly in P116, P279, ‘Seri’ and ‘Silverstar’ by 29%, 25%, 21% and 22%, respectively. Total grain yield per pot was reduced by WSF for all accessions except for 2–49, ‘Seri’ and ‘Silverstar’. The highest reduction in total grain yield was observed for P399 and P279 at 59% and 53%, respectively. Plant height was significantly reduced for most accessions affected by WSF except P116, P396, ‘Seri’ and ‘Silverstar’. The greatest reduction in height was observed for P279 at 31%. WSF significantly reduced leaf chlorophyll content in ‘Silverstar’ and ‘Seri’, which showed reductions of 37% and 39%, respectively. RWC decreased in half of the accessions under WSF treatment. Similar to WSH, WSF treatment caused a reduction for all parameters greater than application of WS or Fusarium alone. Overall, P279 and P399 were the most susceptible to WSF treatment, showing more reduction in growth and yield parameters, while 2–49 was the most resistant.

Table 9. Effect of water stress and inoculation with Fusarium culmorum (WSF) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the stress treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.86ns	0.65*	0.63*	0.71*	0.50*	0.46*	0.64*	0.96ns	0.92ns	0.91ns
Spike weight	0.71*	0.74*	0.70*	0.51*	0.79ns	0.92ns	0.78ns	0.99ns	0.69*	0.74*
Seed/spike	0.68*	0.79*	0.74*	0.50*	0.70*	0.63*	0.70*	0.83ns	0.82*	0.78*
Thousand kernel weight	0.89ns	0.71*	0.93ns	0.75*	0.96ns	0.91ns	0.96ns	0.96ns	0.79*	0.78*
Total grain yield/pot	0.71*	0.49*	0.70*	0.47*	0.64*	0.41*	0.51*	0.93ns	0.89ns	0.85ns
Height	0.80*	0.99ns	0.86*	0.69*	0.94ns	0.87*	0.87*	0.84*	0.99ns	0.93ns
Leaf chlorophyll content	0.89*	0.89*	0.78*	0.82*	0.70*	0.89*	0.75*	0.96ns	0.61*	0.63*
Relative water content	1.00ns	1.00ns	0.89ns	0.99ns	0.73*	0.87*	0.73*	0.88*	0.25*	0.93ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of H. filipjevi combined with F. culmorum (HF) stress

When looking at the interaction between the two pathogens, Fusarium and Heterodera, there were significant interactions for spike weight, seeds/spike and total grain yield, indicating that the accessions do not respond equally to the interaction between the two pathogens (Table 4). As shown in Table 10, the combination of nematode and fungus (HF) reduced final plant numbers for P116, P159, P396 and P399. The highest reduction in final plant numbers was observed in P159 at 42%. A significant reduction in spike weight was observed in P159 and ‘Seri’ (susceptible accession to both Heterodera and Fusarium) under a combination of Heterodera and Fusarium (28% and 18%, respectively). Treatment with HF reduced the seeds per spike in P116 and P159 by 22% and 35%, respectively. TKW decreased significantly in P116 and P159 (18% and 28%, respectively). HF significantly decreased total grain yield per pot in some accessions, reaching reductions of 45% and 39% for P116 and P159, respectively. This was greater than the reductions caused by single applications of Heterodera or Fusarium. Leaf chlorophyll content was reduced by the combination of Heterodera and Fusarium in most accessions. P426 and ‘Seri’ (susceptible to both Heterodera and Fusarium) showed significant reductions in RWC. Overall, P159 and P116 were the most susceptible, while 2–49 was the most resistant to the HF treatment.

Table 10. Effect of inoculation with Heterodera filipjevi and Fusarium culmorum (HF) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the inoculated treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.99ns	0.74*	0.58*	0.92ns	0.79*	0.71*	0.86ns	0.99ns	0.83ns	0.96ns
Spike weight	0.81ns	0.98ns	0.72*	0.99ns	0.97ns	0.99ns	0.86ns	0.93ns	0.82*	0.99ns
Seed/spike	0.86ns	0.78*	0.65*	0.99ns	0.82ns	0.82ns	0.83ns	0.91ns	0.92ns	0.87ns
Thousand kernel weight	0.91ns	0.82*	0.72*	0.96ns	0.98ns	0.93ns	0.99ns	0.96ns	0.89ns	0.94ns
Total grain yield/pot	0.72*	0.55*	0.61*	0.84ns	0.99ns	0.56*	0.82ns	0.88ns	0.85ns	0.98ns
Height	0.92ns	0.98ns	0.97ns	0.98ns	0.99ns	0.94ns	0.99ns	0.97ns	0.99ns	0.96ns
Leaf chlorophyll content	0.93ns	0.84*	0.69*	0.88*	0.80*	0.95ns	0.87*	0.93ns	0.67*	0.83*
Relative water content	0.93ns	0.90ns	0.89ns	0.99ns	0.95ns	0.91ns	0.83*	0.90ns	0.81*	0.91ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Effect of water stress combined with H. filipjevi and F. culmorum (WSHF) stress

When looking at the combined interactions between the two pathogens and drought (a realistic situation that occurs in the field where all stressors occur), there were no significant interactions for any measured parameter, indicating that the two-way interactions (drought × pathogen or pathogen × pathogen) explained the response of the accessions to stress (Table 4). As shown in Table 11, WSHF stress caused a significant reduction in final plant numbers for all accessions except 2–49. The highest reduction (83%) was recorded for P116, while 2–49 showed a slight reduction for final plant number. Spike weight decreased significantly under WSHF, with the highest reduction of 39% observed for P116 and 34% for P159. The WSHF stress significantly reduced the number of seeds per spike as well. The greatest reduction in number of seeds per spike was obtained for P159 at 45%. The WSHF combination significantly reduced TKW in all accessions except for P396 and 2–49, reaching a reduction of 36% in ‘Silverstar’. Total grain yield was also significantly reduced by the WSHF treatment. The highest reduction (77%) was observed for P159, while the lowest reductions were obtained for ‘Seri’ and 2–49 (26% and 30%, respectively). Plant height was significantly reduced, with the greatest reduction of 25% recorded for P52. However, some accessions such as ‘Seri’ and ‘Silverstar’ showed no significant reduction for height under WSHF. Leaf chlorophyll content for all accessions decreased with exposure to the WSHF treatment, with reductions reaching 45%, 46% and 49% in ‘Silverstar’, ‘Seri’ and P426, respectively. Among all accessions, P159, P396, P399, P426 and ‘Seri’ showed a reduction in RWC of 24%, 28%, 8%, 34% and 25%, respectively. A reduction in all parameters due to the WSHF treatment was greater than that observed as a result of water stress, Heterodera or Fusarium separately. Generally, under WSHF, P116 and P159 were the most susceptible while 2–49 was the most resistant.

Table 11. Effect of water stress and inoculation with Heterodera filipjevi and Fusarium culmorum (WSHF) on measured parameters of 10 wheat accessions. Data is expressed as a ratio of the mean of the stressed treatment compared to the mean of the control treatment. Significant differences are based on Fisher’s LSD at (P < 0.05), n = 3

Parameter	P52	P116	P159	P279	P396	P399	P426	2–49	Seri	Silverstar
Final plant No/pot	0.45*	0.17*	0.29*	0.42*	0.38*	0.25*	0.50*	0.91 ns	0.71*	0.36*
Spike weight	0.90 ns	0.61*	0.66*	0.77*	0.82 ns	0.91 ns	0.83 ns	0.82 ns	0.67*	0.71*
Seed/spike	0.78*	0.59*	0.55*	0.76*	0.65*	0.74*	0.64*	0.75*	0.81*	0.75*
Thousand kernel weight	0.85*	0.71*	0.70*	0.85*	0.89 ns	0.77*	0.75*	0.94 ns	0.74*	0.64*
Total grain yield/pot	0.58*	0.28*	0.23*	0.64*	0.50*	0.63*	0.50*	0.70*	0.74*	0.59*
Height	0.75*	0.89*	0.78*	0.81*	0.86*	0.87*	0.83*	0.81*	0.98 ns	0.97 ns
Leaf chlorophyll content	0.74*	0.80*	0.60*	0.68*	0.62*	0.88*	0.51*	0.91*	0.54*	0.55*
Relative water content	0.96 ns	0.98 ns	0.76*	0.98 ns	0.72*	0.92*	0.66*	0.91 ns	0.75*	0.93 ns

*Indicates significances at P < 0.05; ^nsIndicates non-significance.

Disease assessment

Assessment of crown rot caused by F. culmorum under greenhouse conditions revealed that a two-way interaction between F. culmorum and water stress was significant for crown rot, indicating that Fusarium and water stress interacted for crown rot severity (Table 4). Mean comparison of crown rot severity showed that water stress numerically increased crown rot severity of Fusarium (Table 12). In most accession, disease ratings were higher in the water stressed treatments. There was also a significant difference among accessions in every treatment except the treatment combining water stress and both pathogens. This had the highest overall level of disease, and the interaction negated any resistance to Fusarium crown rot in the accessions. In addition, the interaction between Fusarium and accessions was significant for crown rot severity, indicating that the accessions do not respond equally to Fusarium (Table 4). Accessions 2–49 and P426 were the most resistant to Fusarium (Table 12). Similarly, growth room experiments showed that 2–49 and P426 were the most resistant, while ‘Seri’, ‘Silverstar’ and P279 were the most susceptible (Table 14). Assessment of growth parameters and yield components under the Fusraium treatment indicated that P159 and P399 showed the highest reductions, while 2–49 and P426 were the most resistant (Table 6).

Table 12. Crown rot scores on the 10 wheat accessions used in the study according to 1–5 scale in terms of disease development or browning at the main stem base (1: 1–9%, 2: 10–29%, 3: 30–69%, 4:70–89%, 5: 90–99%), as per Erginbas-Orakci et al. (2016). Data within each treatment was analysed with Kruskal-Wallis test, and P values for each treatment given at the end of the column, n = 3

Accession	WSF	WSHF	F	HF
P52	2.66	3.00	2.33	2.33
P116	3.50	3.33	2.83	3.00
P159	3.50	3.06	3.00	4.00
P279	3.33	3.33	2.66	2.70
P396	3.00	3.00	2.33	2.33
P399	3.00	3.33	2.41	2.33
P426	2.00	2.33	1.66	1.66
2–49	1.50	2.33	1.50	1.33
Seri	3.66	3.33	2.66	3.33
Silverstar	3.66	3.33	3.16	2.33
Mean	2.98	3.04	2.45	2.53
P value	0.02301	0.6828	0.04941	0.02944

Abbreviations stand for: WSF = Water stress + Fusarium culmorum; WSHF = Water stress+ Heterodera filipjevi + Fusarium culmorum; F = Fusarium culmorum; HF = Heterodera filipjevi + Fusarium culmorum.

The results from the experiment extracting cysts from pots indicated that a two-way interaction between Heterodera and water stress was significant for cyst density (Table 4). Water stress significantly increased cyst density (Table 13). Furthermore, a significant interaction was observed between Heterodera and Fusarium for cyst density (Table 4). Fusarium inoculation significantly reduced cyst density (Table 13). The interaction between Heterodera and the accessions was significant for cyst density, indicating that the accessions do not respond equally to Heterodera (Table 4). ‘Silverstar’ was the most resistant, while ‘Seri’ and P279 were most susceptible, in terms of extracted cysts per root system (Table 13). Cyst results obtained from the growth room experiments confirmed those of the greenhouse pot experiments (Table 14). Results obtained on yield components and growth parameters showed that ‘Seri’ and P116 were more sensitive, with yield and growth parameters mostly affected under Heterodera stress (Table 7). Accessions P159 and Seri showed significant increases in cyst density, under WSH treatment, of 58% and 77%, respectively, compared with cyst density under Heterodera stress alone (Table 13). In this study, ‘Seri’ and P159 were ranked as susceptible to Heterodera stress and when they were exposed to WSH, their cyst numbers increased. Interestingly, when these accessions were exposed to Heterodera and Fusarium stresses, a high reduction in cyst number (up to 40%) was recorded. The accession ‘Seri’ showed a significant 66% reduction in cyst density under the combination of Fusarium and Heterodera (Table 13). The WSHF treatment caused a 30% increase in cyst density on plants, but this increase was not significant when compared to cyst density under Heterodera stress alone. Among all accessions, only P279 showed a significant increase in cyst density (146%) under the WSHF treatment.

Table 13. Mean comparison of cysts produced by Heterodera filipjevi in 10 wheat accessions under the greenhouse condition. Means with the same letter in each column are not significantly different based on the Duncan test at (P < 0.05), n = 3

Accession	H	WSH	HF	WSHF	Mean
P52	5.16 ± 2.46a	7.33 ± 2.32 bc	3.83 ± 1.31 a	7.16 ± 3.70 bc	5.87 cd
P116	7.00 ± 3.24 a	10.66 ± 0.62 abc	4.00 ± 1.08 a	5.00 ± 5.00 c	6.82 bc
P159	8.00 ± 2.83 a	12.66 ± 2.78 ab	5.66 ± 4.50 a	2.83 ± 0.85 c	7.29 bc
P279	7.50 ± 2.48 a	8.00 ± 0.41 bc	6.83 ± 3.12 a	18.50 ± 9.91 a	10.20 ab
P396	5.00 ± 0.41 a	6.66 ± 1.03 bc	3.66 ± 1.31 a	7.66 ± 5.44 bc	5.75 cd
P399	7.16 ± 0.94 a	8.50 ± 1.78 bc	4.33 ± 4.19 a	7.83 ± 5.78 bc	6.96 bc
P426	8.00 ± 1.78 a	7.33 ± 4.50 bc	1.16 ± 0.85 a	7.33 ± 4.50 bc	5.96 cd
2–49	6.66 ± 1.65 a	7.66 ± 6.28 bc	2.16 ± 2.39 a	11.50 ± 3.89 ab	7.0 bc
Seri	10.33 ± 3.70 a	18.33 ± 6.94 a	3.5 ± 0.82 a	14.16 ± 1.25 ab	11.58 a
Silverstar	2.83 ± 0.62 b	3.33 ± 0.62 c	2.66 ± 0.85 a	2.75 ± 0.75 c	2.91 d

Abbreviations stand for: H = Heterodera filipjevi; WSH = Water stress + Heterodera filipjevi; HF = Heterodera filipjevi + Fusarium culmorum; WSHF = Water stress+ Heterodera filipjevi + Fusarium culmorum. ^±Standard deviation.

Table 14. Mean number of cysts produced by Heterodera filipjevi, and crown rot score of the 10 wheat accessions under the growth room condition

Accession	Crown rot	Reaction	Mean number of cysts	Reaction
P52	2.3 ± 0.15	MR-MS	14 ± 1.58	MS
P116	3.2 ± 0.13	S	21 ± 2.32	S
P159	3.9 ± 0.23	S	20 ± 2.14	S
P279	4.0 ± 0.21	S	15 ± 2.55	MS
P396	2.5 ± 0.17	MR	15 ± 1.32	MS
P399	2.5 ± 0.17	MS	16 ± 2.2	S
P426	2.0 ± 0.00	MR	17 ± 2.29	S
2–49	1.5 ± 0.17	R	21 ± 2.47	S
Seri	3.6 ± 0.16	MS-S	23 ± 2.92	HS
Silverstar	4.5 ± 0.17	HS	3 ± 0.33	R

Abbreviations stand for: R = Resistant; S = Susceptible; MR = Moderately resistant; MS = Moderately susceptible; HS = Highly susceptible.

Growth room assessments

Assessment of crown rot indicated that the accession 2–49 was the most resistant, while P279 was the most susceptible. The highest number of cysts were produced on the highly susceptible accession ‘Seri’ and the lowest number was on the moderately resistant ‘Silverstar’ as shown in Table 14.

Discussion

By including all potential combinations of water stress, Fusarium stress and Heterodera stress on a range of wheat accessions, we have conducted a powerful experiment that allows us to statistically verify the interactions among all these stressors. Looking at a number of growth parameters, several conclusions stand out. First, when considering each stressor separately, it is apparent that there are significant interactions with accessions, indicating that not all accessions responded to these equally. This indicates that there is pathogen resistance and drought resistance among the chosen accessions. When we ask the question, ‘do the two pathogens interact?’, we see a strong indication with spike weight, seeds/spike and total grain yield, in other words, yield parameters. This shows that the combination of the two pathogens causes greater losses than simply the additive effects of each alone. When we ask the question, ‘how does water stress interact with the pathogens?’, we see significant interactions with Fusarium but not with Heterodera. This confirms the extensive literature that crown rot caused by F. culmorum is heavily influenced by drought stress, but Heterodera less so. Finally, when we look at all three stressors combined, we do not see a significant interaction. This indicates that the pathogens and water stress are capable of causing significant losses by themselves, but adding a third stressor does not have a synergistic effect or a greater impact than the additive effects of the stressors.

To date, few studies have been carried out to investigate the interaction between soilborne diseases and plant vulnerability (Atkinson 1892; Jones 1981; Back et al. 2002; Hassan et al. 2012). In this study, we aimed to study the effects of two important soilborne pathogens (nematode and Fusarium) attacking cereal crops, as well as their reactions under drought stress. Interactions among plant-parasitic nematodes and fungi can have complex effects. Plant parasitic nematodes feed on the plant roots and are able to make wounds in the root tissues, ultimately reducing a plant’s ability to absorb water and nutrients from the rhizosphere (Rahi et al. 1988; Audebert et al. 2000; Davis et al. 2014). In addition, roots attacked by plant parasitic nematodes are more susceptible to invasion by other pathogens such as soilborne fungi (Hassan et al. 2012). In this study, it was clear that the single application of the three stress factors, H. filipjevi, F. culmorum and drought, had significant negative effects on agronomic traits, and amplified disease symptoms. This negative effect increased and became more distinct under water stress. These results are in agreement with previously reported results (Papendick and Cook 1974; Beddis and Burgess 1992; Diourte et al. 1995; Audebert et al. 2000; Smiley et al. 2005; Chekali et al. 2011; Ahmadi et al. 2012; Hassan et al. 2012; Scherm et al. 2013).

Seedling death before or after emergence is one of the most typical symptoms of F. culmorum (Scherm et al. 2013). In the present study, seedling emergence significantly decreased with the individual stresses of water stress, F. culmorum and H. filipjevi, and seedling mortality was greater when wheat accessions were subjected to any of the soilborne pathogens combined with water stress, or when more than two stresses were applied. The emergence of 2–49 (resistant to F. culmorum) seedlings was the least affected across all treatments, while emergence of P399 was the most severely affected. This shows the importance of breeding for pathogen resistance to reduce the damage caused by pathogens, in accordance with the earlier report of Chekali et al. (2011). Similarly, co-inoculation of F. udum and H. cajani in pigeonpea plants increased wilt incidence to 100%, while wilt incidence was reduced by 45% and 80% with the fungus and nematode alone, respectively (Sharma and Nene 1989). This is also in agreement with the report of Walker et al. (1998) on M. incognita and T. basicola in cotton crops. Similarly, other studies (Martin et al. 1982; Wheeler et al. 1992; Bowers et al. 1996) showed that the combination of Pratylenchus penetrans and Verticillium dahliae on potato increased early death caused by V. dahliae. Sudden death syndrome (SDS) in soybean caused by Fusarium solani was also enhanced by co-infestation of F. solani and H. glycines, compared with infestation by F. solani alone (McLean and Lawrence 1993).

In this study, the interactions between the pathogens and water stress for the yield components were significant. Reductions in yield components were greater when plants were treated with combinations of stresses than when they were treated with each stress alone. Similarly, co-inoculation of F. culmorum and H. avenae reduced grain yield and TKW (Hassan et al. 2012). In addition, co-occurrence of R. solani and H. avenae led to a greater reduction in wheat grain yield (Meagher et al. 1978). Chekali et al. (2011) reported that F. culmorum inoculation reduced the grain weight of wheat. These findings are in agreement with those of Smiley et al. (2005) and Kane et al. (1987) on Fusarium crown rot.

Similar studies demonstrated that the effects of R. solani and M. javanica on peanuts were greater when the pathogens occurred together compared to with effects of each pathogen alone (Batten and Powell 1971; Golden and Van Gundy 1975; Sankarialingman and McGawley 1994; Walker et al. 1998, and reviewed by Abdel-Momen and Starr 1998). Chekali et al. (2011) indicated that water stress and Fusarium inoculation affected TKW when applied individually, however, no significant interaction was recorded.

The results of the interaction effects showed that water stress had a greater impact on height; when F. culmorum was combined with water stress, however, the reductive effect of water stress increased. The highest reduction was observed under the WSHF treatment. A negative effect of WS on wheat height was reported earlier by Ahmadi et al. (2012). Hassan et al. (2012) also demonstrated that Fusarium or nematodes caused significant reduction in wheat height when applied individually, and that the reduction was greater when they were inoculated simultaneously. Similarly, Chekali et al. (2011) reported that inoculation with F. culmorum caused a significant reduction in plant height (P = 0.005). Moreover, Smiley et al. (2005) reported that crown rot reduced tiller height in wheat. The co-occurrence of drought stress and charcoal rot (caused by M. phaseolina) resulted in a greater reduction in growth parameters, including the accumulation rate of dry matter and leaf area, compared with each stress alone (Mayek-Perez et al. 2002).

In the present study, leaf chlorophyll content was significantly reduced by all treatments, especially when water stress was combined with F. culmorum or H. filipjevi stress. The highest reduction in leaf chlorophyll content was observed in the WSHF treatment. This is in agreement with the results obtained by Nyachiro et al. (2001), Manivannan et al. (2007), Sayar et al. (2008), Lonbani and Arzani (2011) and Farshadfar et al. (2013). Water stress negatively affects cell division and development rates in plants, and consequently reduces vegetative growth (Mayek-Perez et al. 2002). In plants, drought stress induces the production of reactive oxygen species leading to damage of the chloroplasts and a subsequent reduction in chlorophyll (Smirnoff 1995; Iturbe-Ormaetxe et al. 1998). Contrary to these results, Audebert et al. (2000) reported a positive effect of drought and nematode stresses on leaf chlorophyll content in rice. Bauriegel et al. (2011) reported that infection of wheat ears with F. culmorum reduced the efficiency of photosynthesis.

Leaf relative water content (RWC) is an indicator for measuring leaf water status in plants. In the present study, a significant reduction in RWC was observed under all treatments, but the reduction was greatest under WSHF. Similar to our results, Mayek-Perez et al. (2002) observed a negative effect of drought stress on RWC in the common bean, while M. phaseolina had a slight effect in the reduction of RWC. Leaf RWC in drought-tolerant barley cultivars was greater than in drought-sensitive cultivars (Matin et al. 1989). In accordance with our results, H. sacchari accentuated the water stress effect in rice, including a reduction in leaf water potential and stomatal conductance (Audebert et al. 2000).

Roots have a crucial role in soil water absorbance. Cereal cyst nematodes disrupt plant-water relations. CCNs, by causing anatomical changes in the root epidermis, cortex and xylem, form syncytia and subsequently reduce water flow in the vascular cylinder (Shepherd and Huck 1989; Amir and Sinclair 1996; Davis et al. 2014). CCNs also restrict root depth and change the root volume (Amir and Sinclair 1996). Similarly, F. culmorum affects root and crown function by colonizing the root tissues (Beccari et al. 2011), and subsequently reduces water uptake and exacerbates water stress in plants (Kirkpatrick et al. 1991, 1995; Whish et al. 2014). Therefore, a combination of water stress and SBPs accentuates water stress damage to plants.

The results of the present study indicate that germplasm susceptible to F. culmorum had higher reductions in yield components and growth parameters. This was obvious from the results obtained by the Fusarium-resistant accession 2–49, which showed a minimal reduction in yield components and growth parameters. In general, 2–49 had the lowest negative responses in all treatments. Water stress increased crown rot severity and consequently reduced total grain yield and growth parameters. Stress caused by H. filipjevi, on the other hand, showed no significant effect on crown rot when co-occurring with F. culmorum, but increased crown rot severity and reduced total grain yield when applied in triple combination (WSHF). The co-occurrence of water stress and Heterodera increased the cyst density of wheat plants, particularly in accessions susceptible to Heterodera, while co-inoculation of Fusarium and Heterodera reduced cyst density. Accessions susceptible to both pathogens showed a significant reduction in cyst density under co-inoculation with Heterodera and Fusarium. Under a combination of the three stresses (WSHF), cyst density did not significantly increase. Among all accessions, only entries susceptible to both WS and Fusarium, in this case P279, showed a significant increase in cyst density under the WSHF treatment. Fusarium culmorum appears unable to reduce nematode density under the co-occurrence of WSHF at the same level as when it occurs together with HF.

The first study on the interaction between nematode and fungal pathogens was carried out by Atkinson (1892), who reported that root-knot nematodes (Meloidogyne spp.) increased wilt disease in cotton (caused by Fusarium oxysporum f.sp. vasinfectum). In agreement with our results, Hassan et al. (2012) reported that pre-inoculation of wheat with H. avenae one month before inoculation with F. culmorum accentuated the infection severity of the fungus. Back et al. (2002) reviewed the evidence for how co-infection of fungus and nematode increase fungus severity. Cyst nematode juveniles penetrate root tips and migrate intracellularly to the vascular cylinder (Von Mende et al. 1998). Soilborne fungi have also been found to exploit nematode invasion sites to penetrate underlying tissue (Orion et al. 1999). The modification of root cells by nematodes, which overcomes chemical barriers to allow penetration of the cortex, may predispose plants to root-infecting pathogens (Shepherd and Huck 1989). Cyst nematodes such as those of the Heteroderae family form feeding sites (syncytia) in the roots, which contain higher levels of total protein, amino acids, lipids, DNA and sugars for the duration of their development and reproduction (Taylor 1990; Abawi and Chen 1998). Therefore, one hypothesis is that these nutrient-dense cells are beneficial for colonizing fungi (Jones 1981; McLean and Lawrence 1993; Abdel-Momen and Starr 1998).

In accordance with our results, the interaction between H. avenae and F. culmorum (Hassan et al. 2012), Trichoderma harzianum and H. avenae (Ibrahim et al. 1997), F. solani and H. glycines (McLean and Lawrence 1993), Gaeumannomyces graminis and H. avenae (Cook 1975), F. udum and H. cajani (Sharma and Nene 1989), and H. schachtii and Verticillium wilt reduced the nematode cyst population.

A combination of diseases likely makes plants more prone to early senescence and death, and consequently may impede nematodes from completing their life cycle, thereby reducing nematode density (Hassan et al. 2012). Fungi penetrate nematode eggs by hyphae through mechanical or chemical mechanisms. Hyphal growth depends on the consumption of nutritional resources in the nematode eggs (Ashrafi et al. 2017). One more reason for the reduction of nematode density when co-inoculated with fungi may be competition of pathogens for nutrients in the root zone (Hassan et al. 2012), or the interference of the fungus causing root rot in the nematode developmental cycle (McLean and Lawrence 1993).

The results observed with respect to disease severity under water stress are in agreement with those reported by Chekali et al. (2011), who showed that water stress significantly accelerated disease severity caused by F. culmorum. Diourte et al. (1995) and Mayek-Perez et al. (2002) reported similar findings where drought stress in sorghum and common bean increased susceptibility to Macrophomina phaseolina (charcoal rot fungus). Beddis and Burgess (1992), in a study of the influence of WS on infection and colonization of wheat seedlings by Fusarium graminearum group 1 (Fusarium pseudograminearum), revealed that seedlings under WS were more susceptible to disease development than seedlings under non-stressed conditions. Papendick and Cook (1974) indicated that dryland conditions accentuated foot rot in winter wheat. Similarly, charcoal rot symptoms were more severe under drought stress in the common bean, as reported by Mayek-Perez et al. (2002).

In agreement with our results, Chekali et al. (2011), Papendick and Cook (1974) and Beddis and Burgess (1992) suggested that higher susceptibility to pathogens in water stress-treated plants is probably related to the disruptive role of low water potential in physiological processes such as plant defence mechanisms. It appears that water stress affects host susceptibility to pathogens, rather than pathogen development. Drought stress can affect plant resistance to pathogens in a detrimental manner (Atkinson and Urwin 2012). Audebert et al. (2000) showed that drought stress reduced the density of H. sacchari in rice.

Plants have evolved some defence mechanisms to protect themselves from pathogen attacks, such as plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). These hormones play a central signalling role and are involved in regulating defence responses (Jiang et al. 2010). Absicic acid (ABA) is an important plant hormone that plays a signalling role in responses to various abiotic stresses like drought stress. ABA signalling pathways suppress both SA and JA-ethylene signalling pathways, so co-occurrence of drought and diseases may enhanced susceptibility to pathogens (Anderson et al. 2004).

In conclusion, this study shows that there is a significant interaction among biotic and abiotic stresses, which exacerbates their individual disruptive effects. One of the most significant effects caused by the combination of these three stresses was the high seedling mortality. The lowest level of damage was observed when a host accession resistant to F. culmorum was used. In addition, accessions resistant to F. culmorum showed greater stability in yield components under water stress. This study underscores the importance of wheat breeding to develop resistant germplasm. Breeders, mycologists, agronomists, physiologists, and nematologists should work together to mitigate crop losses caused by the interaction of multiple pathogens, particularly when these occur under drought conditions.

Acknowledgements

Thanks go to Dr Marta Lopez for providing the Iranian germplasm. Special thanks to the Transitional Agricultural Research Institute (TZARI), Eskisehir for the technical support to the PhD student (Mahin Ahmadi). The authors would like to thank CIMMYT International for funding this study

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by International Maize and Wheat Improvement Center (CIMMYT) Turkey