Varieties of ornamental sunflower and inoculants of arbuscular mycorrhizal fungi

Four varieties of ornamental sunflower were used: belleza de otoño (BO), doble gigante (DG), doble enana (DE), and girasol simple amarillo (GSA) of the company Vita⁽ (Morelos, MX). The seeds were washed with sodium hypochlorite (5%) for five min and subsequently rinsed thrice with distilled water. Then, seeds were placed in absorbent paper moistened with tap water following the technique “tacos de germinación” (germination wraps) (^{Moreno, 1996}). These wraps were maintained in darkness conditions at 25 (C for five days. Subsequently, seedlings were transplanted to pots, selecting those of approximately 6 cm in height. The AMF inoculants used were: Rhizophagus intraradices (Ri), Funneliformis mosseae (Fm), and native consortia of agave (Agave cupreata) rhizosphere: Cerro del Metate (CM) and Las Campesinas (LC) described by ^{Trinidad et al. (2017)}. These inoculants were previously propagated for eight months under greenhouse conditions and in pot traps using sorghum (Sorghum bicolor ( Sorghum sudanese Sweet Chow; Western Seed Co.; Kitale, KE) as host. Sunflower varieties were inoculated with 100 AMF spores at the moment of transplanting and placed directly on the root. The substrate used in the experiment was 50:50 (v/v) of Peat Moss (Sunshine Mix⁽ No. 3, Agawam, USA) and a soil:sand:agrolite (60:25:5, v/v/v) mixture, which was sterilized in autoclave (120 (C, 1.05 kg m^-2, 6 h). Black 5 kg polyethylene bags were used as pots for one sunflower plant, placing 3 kg of sterilized mixture in each bag.

Environmental conditions during the experiment

The experiment was performed in field conditions in the facilities of Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) at Zapopan located at 20° 42’ 5.97” N, 03° 28’ 25.83” W in the rainy season (14 June to 23 August) of 2017. The average temperature was 20.3 (C with an average maximum of 31 (C and minimum of 15.5 (C. During the experiment a heavy accumulated precipitation of 279.1 mm was recorded at an average wind speed of 2.1 km h^-1 (^{IAM-CUCEI, 2017}).

Evaluation assay of arbuscular mycorrhizal fungi in ornamental sunflower growth

A bifactorial 5 × 4 randomized complete block design was used with six replicates per treatment. Blocking eliminated the solar radiation factor. The experimental unit was a pot with a sunflower plant. The five levels of the first AMF factor were (1) Rhizophagus intraradices (Ri); (2) Funneliformis mosseae (Fm); (3) Cerro del Metate (CM); (4) Las Campesinas (LC); (5) without-AMF (control). The four levels of the second ornamental sunflower factor were (1) belleza de otoño (BO); (2) gigante simple amarillo (GSA); (3) doble enana (DE); (4) doble gigante (DG). At the end of the experiment (70 days after transplant), plant height, stem diameter, leaf number, floral chapter diameter, dry root weight, stem with leaves, floral chapter and mycorrhizal colonization were quantified in each experimental unit. For mycorrhizal colonization, the clearing and staining technique developed by ^{Phillips and Hayman (1970)} was followed; for quantification, the method proposed by ^{Hernández-Cuevas et al. (2012)} was used, which consisted of taking 90 stained roots of 2 cm in length; 30 roots were placed per slide in a compound microscope (40X) to observe the mycorrhizal symbiosis structures; the mycorrhizal colonization percentage (MCP) was estimated by the following formula: MCP = [(Number of colonized fields/Number of total fields observed) ( (100)]. The data obtained in the assessment of the different response variables were subjected to an analysis of variance (ANOVA) and a Tukeys’s multiple comparison of means tests (P ≤ 0.05). Dry weight data were transformed with a natural logarithm; for dry weight and mycorrhizal colonization, Pearson’s correlation analysis was carried out. These statistical analyses were performed with the Statgraphics Centurion (^{StatPoint Inc., 2005}) program.

Sunflower growth in association with arbuscular mycorrhizal fungi depends on plant variety

In general, 70 days after the experiment was established, the plants of the variety gigante simple amarillo (GSA), both inoculated and without-AMF inoculation showed greater growth compared with the other varieties. The treatment sunflower-GSA inoculated with Cerro del Metate (CM) showed significantly greater plant growth (Tukey’s, P ≤ 0.05) when compared with the variety GSA without-AMF. The inoculant CM promoted plant growth of the variety GSA in the majority of the response variables, except for height and leaf number (Tukey’s, P ≤ 0.05) (Tables 1 and 2).

The ornamental sunflower varieties showed a wide plasticity response to inoculation with native mexican arbuscular mycorrhizal fungi

For the ornamental sunflower variety factor, plant growth, both those inoculated and without-AMF, the majority of the response variables followed this order: GSA > DE > DG > BO. This tendency suggested a differential response due to the intrinsic genotype of each variety; under this idea, the varieties GSA and BO showed the greatest and least plant growth, respectively (Table 3). With respect to the response of the sunflower varieties to mycorrhizal colonization, a tendency GSA = DG = DE > BO was observed, which showed that all the varieties were capable of establishing the mycorrhizal association; however, the degree by which they were colonized was variable (Table 3). For the AMF inoculant factor, observing the growth variables with significant differences (Tukey’s, P ≤ 0.05) a tendency of without-AMF = Fm > CM = Ri > LC could be established, which suggested that AMF have negative effects in ornamental sunflower plant growth. Under this context, the LC consortium was the inoculant with the greatest negative effect, followed by CM and Ri; whereas Fm did not show any effect on growth when compared with the control group without-AMF sunflower plants (Table 3) while for mycorrhizal colonization the following order was observed Ri = CM = LC > Fm = without-AMF.

These results show that the Fm inoculant colonized ornamental sunflower variety little and did not cause negative effects in their growth (Table 3), which could be due to the existing interaction among the sunflower varieties and AMF inoculants in the different response variables assessed except for leaf number and mycorrhizal colonization (P ≤ 0.05). This result suggested that the sunflower varieties responded differently to the different AMF inoculants (Table 3, Figure 1), showing a wide plasticity response of the ornamental sunflower varieties to mycorrhization. These tendencies are clearly observed in the data shown in Table 4, where the significantly (Tukey’s, P ≤ 0.05) positive effects of the different AMF inoculants can be appreciated in each one of the sunflower varieties; for example, an increase of 31% by the CM inoculant observed in the GSA variety with respect to the group without-AMF inoculated treatment, and even negative effects since in the BO variety all the AMF inoculants showed a decrease in growth (Figure 1, Tables 4 and 5).

Sunflower varieties: GSA = gigante doble amarilla; DE = doble enana; DG = doble gigante; BO = belleza de otoño. Inoculants of AMF: Ri = Rhizophagus intraradices; Fm = Funneliformis mosseae; consortia Cerro del Metate (CM) and Las Campesinas (LC).

Figure 1: Interactions among inoculant factors of arbuscular mycorrhizal fungi (AMF) and ornamental sunflower varieties in pot at open sky at 70 days after transplant (rainy season 2017; Zapopan, Jalisco).  

Mycorrhizal colonization and plant growth are related in the belleza de otoño (BO) variety

The correlation analysis showed an r value of -0.4078 between dry weight and mycorrhizal colonization for the BO variety, which revealed that while mycorrhizal colonization percentage increased, plant growth was lower in the BO sunflower variety. This effect was not observed in the rest of the sunflower varieties, for which the (r) correlation values were statistically zero (P > 0.05); this result suggested that no relationship existed between mycorrhizal colonization and plant dry weight (Table 5).

Discussion

The sunflower showed a great plasticity in plant growth by the effect of mycorrhizal colonization; in this study GSA was stimulated by CM and inhibited by LC while no significant effect was found with any AMF for the DE variety (Tukey’s, P ≤ 0.05); in contrast, BO growth was inhibted by all AMF inoculants whereas for DG, the effect in growth was negative with LC. The high response variability (from parasitic to mutualistic) of different plants in the presence of mycorrhizal fungus strains is a phenomenon reported by ^{Klironomos (2003)} where magnitude and direction were determined by the combination plant-fungus. Additionally, no variety showed its best effect with the same mycorrhizal inoculant. This study has shown that the plasticity response to AMF is not only due to the differences among species but also to the varieties of the same plant species. The responses the sunflower showed to mycorrhizal colonization, in terms of growth, depended on the balance between the organic carbon donated to the fungus and phosphorus absorption that it provided to the plant (^{Smith and Smith, 2012}). A positive growth effect is attributed to the adequate phosphorus absorption of the plant through the AMF, and a negative response is attributed to this same fault of the fungus to provide phosphorus at the same time it consumes too much carbon (^{Smith and Smith, 2012}).

A mycorrhizal fungus may be considered as a plant parasite when the net cost of symbiosis exceeds the net benefit. Parasitism may be induced by the plant development by the ambience or inclusive when determined by the plant species genotype (^{Johnson et al., 1997}). For example, the lack of light or phosphorus fertilization is considered a condition that favors parasitism by the fungi that form mycorrhizae (^{Friede et al., 2016}) and cause mycorrhized plants to grow less than those non-mycorrhized (^{Daft and El-Giahmi, 1978}; ^{Ballhorn et al., 2016}). Nevertheless, ^{Friede et al. (2016)} observed that Hieracium pilosella (obliged mycotrophic) formed mutualistic type mycorrhizal associations consistent under edaphic and soil conditions, both promoters of mutualism and parasitism, while Corynephorus canescens (less mycotrophic) showed a pronounced parasitism association in all the cases. These results demonstrated that the host genotype had an important function in mycorrhizal symbiosis. In this case, Belleza de Otoño was consistently affected by mycorrhization (independently of the inoculum type), which suggested it was a genotype effect.