Colonization dynamics of arbuscular mycorrhizal fungi (AMF) in Ilex paraguariensis crops: Seasonality and influence of management practices

1 Introduction

The yerba mate, Ilex paraguarienses St.-Hill. (family Aquifoliaceae) is a species originally from South America, native to subtropical regions of Argentina, Brazil, Paraguay and Uruguay. It is native to the phytogeographic region of Selva Paranaense of the Brazilian Atlantic Rainforest. Argentina is the main manufacturer and consumer of yerba mate worldwide, with 200,000 implemented hectares and 17,400 producers distributed in Misiones and NE of Corrientes, being the utmost important socio-economic crop at the regional level (INYM, 2011).

Mycorrhizal associations exert great influence on diverse agroecological and ecosystemic processes. Currently, there is a growing awareness of soil biodiversity and of the potential of these natural resources as alternative or complementary technologies to agrochemicals. Inoculation with arbuscular mycorrhizal fungi (AMF) (phylum Mucoromycota; Spatafora et al. 2016) is an interesting strategy for improving crop yields because arbuscular mycorrhizal symbiosis provides numerous services to crops (Gianinazzi et al., 2010), including efficient use of fertilizers and soil nutrients (Javaid, 2009), protection against drought stress (Porcel et al., 2007; Porcel and Ruiz-Lozano, 2004) and diseases (Liu et al., 2007), increased N-fixation in legumes (Barea and Azcon-Aguilar, 1983; Haselwandter and Bowen, 1996), and improved soil physical properties (Hallett et al., 2009). However, since AMF are obligate biotrophs that require a host plant to complete their life cycle (Smith and Read, 2008). They colonize the root tissues through sources of propagules existing in the soil, including mature spores, mycorrhized root fragments or mycorrhized plants that grow in their vicinity. AMF hyphae generally do not have septa and may grow either extra or intraradically. Intraradical mycelium produces characteristically branched structures inside the cortical cells called arbuscules. Many AMF species also form large globular intraradical cells called vesicles that have a reserve function. Although arbuscules are considered diagnostic structures of arbuscular mycorrhizal symbiosis, there is a wide range of other intraradical structures formed by AMF, including intracellular hyphal coils, which sometimes occur without the presence of arbuscules. The spores of AMF are asexual, multinucleated and produced directly by the mycelium, both inside and outside the root (Smith and Read, 2008).

Many studies worldwide have focused on the use of AMF in sustainable agriculture as a mechanism to maintain efficient commercial crops (Andrews et al., 2012; Pellegrino et al., 2011). There are also countless studies on this subject for tropical and subtropical crops (e.g. coffee, cacao, avocado, banana, pineapple, citrus) and forest trees, which showed the positive effects of mycorrhization on their biometric characteristics (Adriano-Anaya et al., 2011; Aguirre-Medina et al., 2011; Colozzi-Filho-Siqueira, 1986; da Silveira et al., 2003; de Oliveira and de Olivera, 2005). To add there are few rigorous studies that hither to established the degree of yerba mate association with AMF, especially under field conditions. Andrade et al. (2000) reported the presence of arbuscules, coils, appressoria, intracellular aseptate hyphae, and vesicles in roots of I. paraguarensis in Santa Catarina, Brazil

In Argentina, more than 60% of the area cultivated with yerba mate has serious problems of soil degradation. Its recovery demands of already developed technologies, and of research and technological innovations of existing tools where AMF would play an important role. In a survey of yerba mate seedlings from 10 municipalities in the States of Paraná and Santa Catarina, Gaiad and Lopes (1980) registered that roots from 90% of the samples showed some degree of mycorrhizal association. The aim of the present study was to determine the influence of management practices and seasonality on root colonization on plants of yerba mate.

2 Materials and methods

3 Results

3.2

3.2 Arbuscular micorrhizal fungi colonization

Mycorrhizal structures associated with root colonization in yerba mate were internal hyphae, vesicles and arbuscules.

We evaluated the total percentage of colonization and internal structures of AMF in yerba mate plants according to the sites, seasonality, years and the interaction between them.

The percentages of total mycorrhizal colonization and mycorrhizal structures associated with root colonized (arbuscules, internal hyphae and vesicles) in different sites are shown in Fig. 1. Significant differences among the six sites were observed, with the highest values registred in NAT and the lowest values in Y28 and CU.

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Fig. 1

Percentage of root colonized (arbuscules, hyphae internal, vesicles, and total colonization) in yerba mate crop at the six sites analyzed. Values are means for the sampling sites during 2013 and 2014. The same letter above the bars indicates that values do not differ significantly by one-way ANOVA and Tukey’s test (p < 0.05). References: L49 (Loma 49), CN (Carmelo Nuevo), CU (Colonia Unión), Y24 (Yerbal 24), Yerbal 28 (Y28), and NAT (native plants).

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When analyzing the effect of seasonality, significant differences in the total colonization between summer and winter were found (p < 0.05). Higher percentages of colonization were registered in winter than in summer (67.5% y 42% respectively). Total colonization also shown significant differences between years. A 49,5% of colonization was registered in the year 2013, and 60% in 2014. Significant interactions for season-sites were found (p < 0.05) in the percentages of total mycorrhizal colonization (Table 4).

Table 4 Analysis of variance for the percentage of total AMF colonization and mycorrhizal structures (internal hyphae, vesicles and arbuscules) in plants of yerba mate during summer and winter at the sampling sites analyzed. Values with (*) are significantly different (Tukey’s test, p < 0.05).

	Internal hyphae			Vesicles			Arbuscules			Total colonization
	df	F	P	df	F	P	df	F	P	df	F	P
Sites	5	3.72	<0.05*	5	1.17	0.33	5	2.42	<0.05*	5	1.68	<0.05*
Season	1	11.94	<0.05*	1	2.77	0.1	1	10.01	<0.05*	1	10.0	<0.05*
Sites x Season	5	5	<0.05*	5	2.85	<0.05*	5	1.61	0.17	5	2.43	<0.05*

When considering only the percentages of internal hyphae and arbuscules, significant differences between sampling sites were observed, being the highest values found at NAT and Y24 and the lowest at CU and Y28. Almost all mycorrhizal structures except the percentage of vesicles showed significant differences between seasons and between sites. Significant interactions for season-sites were found in internal hyphae and vesicles.

The percentage of total mycorrhizal colonization was significantly correlated (p < 0.05) with the soil content of organic matter (r = 0.92) and N (r = 0.88)

4 Discussion

This study confirms the association of I. paraguariensis with AMF under different crop situations and in native plants growing under natural conditions. Bergottini et al. (2017) have analyzed the diversity of the root-associated microbiome of this crop. Also, there are many studies in subtropical and tropical species of fruit and forest trees, such as coffee (Aguirre-Medina et al., 2011; Montilla et al., 2005), cacao (Aguirre-Medina et al., 2007) and avocado (da Silveira et al., 2003; Montañez Orozco et al., 2010), which showed similar values of colonization to those obtained in the present work. Root colonization by AMF under field conditions is determined by several aspects, such as soil physical and chemical factors (pH, temperature, aeration, texture and P and organic matter contents), climate (intensity and duration of light) and crop management (soil preparation, application of agrochemicals and cultural practices) (Pérez and Vertel, 2010).

Our results showed higher mycorrhizal colonization in NAT than in cultivated sites. The lowest colonization values were registered in Y28 and CU, which are the sites with higher intensity of tillage. The history of culture of a particular site is very relevant to encourage the early establishment of a mycorrhizal colonization and capitalize before its benefits. Soils without tillage or with reduced intensity are appropriate to ensure the persistence of a diverse population of AMF in the soil and maintain the integrity of the extraradical mycelium (Goss et al., 2017). Colonization from an intact extraradical mycelium occurs earlier and develops more rapidly than from spores, colonized root segments or fragmented hyphae. Therefore, it is considered that AMF colonization is more effective starting from an intact extraradical mycelium. Karasawa et al. (2001) indicated that the potential inoculum in soil and colonization capability remains more constant in natural ecosystems than in agricultural systems. Agricultural practices are a source of disturbance that affect both dynamics and diversity of AMF, lowering the propagules density and colonization capability of soils.

Considering the sites under cultivation, the higher percentage of colonization was found at Y24. In this site, a sustainable management is used which allows to maintain the soil microbiota undisturbed. Hawkins and George (2001) reported that in systems with no inputs, the propagule bank in the soil remains diverse and active. In this way, mycorrhizal association is favored, as is shown in this study.

Our study shows that the percentage of AMF colonization correlated significantly with organic matter and N contents. The highest organic matter and N contents, characteristic of rainforest environments, were found in the soil samples from NAT and correlated significantly with the percentage of AMF colonization. Jodice and Nappi (1987) reported that the content of soil organic matter positively influenced AMF activity. Regarding N, Chen et al. (2014) considered that AMF colonization is not influenced by the concentration of this nutrient in soil. However, it has also been frequently observed that the effect of N concentrations on AMF colonization depends to a large extent on particular conditions of each crop and consequently, no clear pattern can be predicted (Chen et al., 2014; Treseder 2004).

In addition to the influence of soil type and management practices, it is important to consider the influence of seasonality. In this study, a higher percentage of colonization was found in winter (lower physiological activity). Bohrer et al. (2004) indicated that seasonality has minimum influence on arbuscular mycorrhizal colonization, and that it should be explained through host plant phenology. According to this, Mullen and Schmid (1993) confirmed that, in some species, higher values of colonization occurs when the plant needs to accumulate P, before the season of maximum physiological activity.

Differences found in colonization between years 2013–2014 can be explained by the rainfalls, since temperature variation between 2013–2014 was not registered in the sampling sites. To analyze the relationship between AMF colonization and rainfall, not only the annual rainfall but also its distribution should be considered, particularly of rainfall that occurred in the four months prior to sampling. Due to the type of soil and to the way in which the yerba mate is grown, it is considered that under field conditions, the vegetative state of the crop at a given time better reflects the water status during the last three to four months than the total annual rainfall. For this reason, we considered the rainfall that occurred in May, June, July and August for the winter sampling, while for the summer sampling, the rainfall that occurred in September, October, November and December. In this study, AMF colonization was positively correlated with rainfall. At NAT (Misiones), rainfall during 2014 was 89% higher than in 2013. These differences were correlated (r = 0.94, data not shown) with an increase in colonization of 19% during the rainiest year. The same pattern was observed for the cultivated sites Y28, Y24, CU, CN and L49 (Corrientes), where it was registered 45% more rainfall in 2014 than in 2013 and an increase in colonization of 12%. These findings are in agreement with those reported by other authors (Apple et al., 2005; Becerra et al., 2007; de Oliveira and Oliveira, 2005), who found that in well-drained soils the highest percentage of AMF colonization occurred during the rainy season. Newhall and Berdanier (1996) pointed out that roots are subjected to strong hydric and nutritional stresses related to changes in soil water content, which negatively affect mycorrhizal colonization.

This work confirms the association of I. paraguariensis with AMF, showing that yerba mate is a host species under both crop and natural conditions. The results also show that the percentages of arbuscular colonization decrease when the tillage practices are more intense. Considering the current progress on the knowledge of mycorrhizal associations in crops of yerba mate, it is necessary to develop a methodology aimed at protecting and maximizing the benefits of the symbiosis with these microorganisms that are naturally and evolutionary associated with yerba mate.