Anatomophysiological modifications induced by solid agricultural waste ( vermicompost ) in lettuce seedlings ( Lactuca sativa L . )

Resumen. El objetivo de este trabajo fue analizar el impacto del tratamiento de vermicompuesto en plántulas de lechuga en términos de modificaciones anatómicas y fisiológicas relacionadas a la partición de asimilados y crecimiento. Los resultados mostraron que el efecto de vermicompuesto en el aumento de crecimiento se explica por un aumento en la actividad del meristema fundamental de la hoja. Se observó en la hoja un mayor espesor y número de capas de clorénquima. Esto se relaciona con un incremento en la actividad fotosintética, expresado por un aumento en la Tasa de Asimilación Neta. El vermicompuesto también actuó a nivel de procambium, produciendo un aumento en el número de miembros de vasos y en el área de floema, lo que está vinculado a una mayor eficiencia en la transferencia de fotoasimilados. Este hallazgo está relacionado a un menor coeficiente de área foliar efectiva en el tratamiento con vermicompuesto, lo que indica una mayor eficiencia de producción y transferencia de fotoasimilados. La evidencia experimental presentada muestra que el vermicompuesto actuó a nivel de meristema fundamental y procambium, produciendo modificaciones anatómicas que incrementaron la biomasa y modificaron la distribución de fotoasimilados y consecuentemente el crecimiento de las plantas.

Abstract.e objective of this work was to analyze the impact of a vermicompost treatment on anatomical and physiological modi cations related to assimilate partitioning and growth in lettuce seedlings.e results showed that vermicompost increased growth, which was most likely due to an increased activity of the ground meristem of the leaf blade.A greater height and number of chlorenchyma layers were observed in the leaf blade. is was related to an increase in the photosynthetic activity, expressed by an increase in the net assimilation rate.Vermicompost also showed an e ect at the procambium level, producing an increase in the number of vessel members, and in the phloem area, which was related to a greater e ciency in the transfer of photoassimilates. is nding was connected with lower E ective Leaf Area Coe cients in the vermicompost treatment, which indicated the greater production efciency and transfer of photoassimilates.e experimental evidence presented here showed that vermicompost showed e ects at the levels of the ground meristem and procambium, producing anatomical modi cations that increased biomass, and improved the distribution of photoassimilates and, consequently, the growth of plants under treatment.

INTRODUCTION
As an alternative fertilization method in response to environmental concerns, the recycling of Solid Agricultural Waste has become known through the production of a biofertilizer generated by earthworms (Eisenia foetida).This vermicompost is traditionally used as an organic amendment.Although this is an old concept in agronomy, vermicompost is a highly useful and viable technological resource for tackling current environmental concerns.It is well-established that earthworms have beneficial physical, biological and chemical effects on soils, and many researchers have demonstrated that these effects can increase plant growth and crop yield (Edwards, 1998;Atiyeh et al., 2000;Argüello et al., 2006).
Previous studies conducted at the Laboratory of Plant Physiology (Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina) showed that vermicompost stimulates lettuce seedling growth and strength (Ledesma et al., 2001).It also improves the economy of water and carbon, maximizing growth and even strengthening transplanting.In garlic, the use of vermicompost produces an earlier start of bulbification and increases the assimilate partitioning index (Argüello et al., 2006).
Vermicompost application to lettuce seedlings significantly improved growth (Atiyeh et al., 2002), increased lettuce both fresh and dry biomass weights (Ali et al., 2007), and the enhanced plant weights were independent of the nutrient content of the substrates.In other crops, this biofertilizer greatly increased foliar area and biomass (Lima & Silva, 1998).Treatments involving vermicompost generally stimulate growth.Atiyeh et al. (2002) also mention a number of references in the literature showing that plant growth regulators, such as auxins, gibberellins and cytokinins, are produced by microorganisms.It has been suggested that the promotion of microbial activity in organic matter by earthworms may result in the production of significant quantities of plant growth regulators (Krishnamoorthy & Vajranabhiah, 1986;Tomati et al., 1983Tomati et al., , 1988Tomati et al., , 1990;;Tomati & Galli, 1995;Edwards, 1998).Earthworm activity accelerates the humification of organic matter, and its influence in increasing microbial populations enhances the presence of auxins and gibberellin-like substances as well as humic acids (Casenave de Sanfilippo et al., 1990).Similar results were presented in biosolids by Zhang et al. (2009).
From an anatomical point of view, it has been determined that the primary growth of plants has its origin at the level of the ground meristem, procambium and protoderm (Dickinson, 2000;Evert, 2008).Mineral nutrition contributes to structural organization, since anatomical modifications are found when plants receive fertilizers, which can alter tissue thickness (Marschner, 1995).Anatomical studies in coffee to determine the effects of nutrients on anatomy have shown that modifications induced in tissues can also influence assimilate partitioning (Rosolem & Leitte, 2007).However, there is no information in the literature to date that explain how vermicompost affects the anatomy and physiology of lettuce seedlings, and impacts assimilate partitioning (Marschner, 1995).
The xylem is the tissue that transports water and minerals from the root system to the aerial portions of the plant, and the phloem translocates the products of photosynthesis from mature leaves to areas of growth and storage, including the roots.The photoassimilates move from the production zones, called sources, to metabolism or storage zones, called sinks.A mature leaf is capable of producing photosynthates in excess of its own needs (Taiz & Zeiger, 1998) Our hypothesis is that vermicompost stimulates lettuce plantlet growth acting at the ground meristem and procambium levels of the shoot, and also optimizes physiological aspects, such as biomass increase and assimilate partitioning.
The aim of this study was to analyze the impact of vermicompost on lettuce seedlings as regards anatomo-physiological modifications related to assimilate partitioning and growth.

MATERIALS AND METHODS
Plant material.Young lettuce (Lactuca sativa L.) var.Criolla Verde plants were grown under greenhouse conditions at the Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina.The greenhouse conditions were: temperature between 20 and 25 °C, relative humidity of approximately 70% (± 20%), and natural light.The growth medium was soil (Entic haplustoll) with and without vermicompost taken from solid waste from industry fridge (All Green Company).
Treatments were (a) soil control (C) and (b) 1 soil: 1 vermicompost (by volume) (V).The physicochemical parameters evaluated were organic carbon, phosphorus, total nitrogen, electrical conductivity and pH.The organic C content was determined by Walkley-Black (Nelson and Sommers, 1996).Total N was evaluated by Kjeldahl and extractable P by Bray and Kurtz Nº1 (Kuo, 1996).The pH values were measured in aqueous extracts 1:2.5 with a pH meter (Orion Research 901).Electrical conductivity was measured in saturated paste with a conductivity meter (DIST4 of HANNA Instrumental).

DISCUSSION
Previous studies demonstrated that lettuce seedlings treated with vermicompost showed greater vigour and growth (Ledesma et al., 2001); the experiments reported here gave similar results.One possible explanation for this is that a significant increase in aerial biomass may be generated with a consequent increase in photosynthesis (Fig. 1 A).Other authors have also found increases in biomass and foliar area in lettuce (Sganzerla, 1983;Mujahid & Gupta, 2010), in tomatoes (Atiyeh et al., 2002) and coriander (Lima & Silva, 1998).This is explained in terms of the increase in NAR (Fig. 1 B), which is related to a lower FAC in the treatment with vermicompost, indicating greater efficiency in the production of photoassimilates (Fig. 2 A).The present investigation demostrated that this increase in NAR is due to an increase of approximately 50% in the number of cell layers and in mesophyll height (Fig. 3: A-3, B-3 and Fig. 4).This increase can most probably be explained by the effect of vermicompost on the number of mesophyll layers, increasing the NAR.
In this context, it is worth asking what plants treated with vermicompost do with their greater production of photoassimilates.Previous findings indicate that vermicompost changes the assimilate partitioning pattern, prioritizing distribution to the aerial part in lettuce (Ledesma et al., 2001), and to the bulb in garlic (Argüello et al., 2006).
How then can this greater distribution be explained from the anatomical point of view?The results suggest that vermicompost acts to increase the number of vessel members (Fig. 3: B-1 and Fig. 4 C) and the phloem area (Fig. 4 D), and it also seems clear from the above discussion that vermicompost increases procambium activity.These anatomical modifications are consistent with a greater efficiency in the assimilate partitioning, which is seen in a decrease in the FAC and SLA Coefficients (Fig. 2 A-B), which in turn, accounts for the greater Harvest Index (Fig. 2

C).
A deeper analysis of Figure 4 (A-B) reveals that the ground meristem activity is manifested in the vermicompost treatment by an increase (1) in the number of mesophyll layers at 30 days after sowing, and (2) of the mesophyll thickness at 45 days after sowing.On the other hand, it is important to point out the increase in the number of vessel elements (Fig. 3A and 4C) and phloem area (Fig. 3B and  4D) which occured after 15 days from sowing, as a result of the same treatment.This can be linked to anticipation in the activity of the procambium.These might be explained by the presence of growth hormones in the vermicompost, wich are absorbed in humic acids, forming complexes (Atiyeh et al., 2000(Atiyeh et al., , 2002;;Arancon et al., 2004Arancon et al., , 2006;;Edwards et al;2006).The hormones present constitute an external "signal" that acts on the primary meristems mentioned earlier, which are responsible for the perception of signals that stimulate growth (Argüello et al., 2008).
In conclusion, experimental evidence suggest that the vermicompost increases growth showing a greater generation and translocation of photoassimilates, which can be explained in terms of various anatomical modifications found.

INTRODUCTION
As an alternative fertilization method in response to environmental concerns, the recycling of Solid Agricultural Waste has become known through the production of a biofertilizer generated by earthworms (Eisenia foetida).is vermicompost is traditionally used as an organic amendment.Although this is an old concept in agronomy, vermicompost is a highly useful and viable technological resource for tackling current environmental concerns.It is well-established that earthworms have bene cial physical, biological and chemical e ects on soils, and many researchers have demonstrated that these e ects can increase plant growth and crop yield (Edwards, 1998;Atiyeh et al., 2000;Argüello et al., 2006).
Previous studies conducted at the Laboratory of Plant Physiology (Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina) showed that vermicompost stimulates lettuce seedling growth and strength (Ledesma et al., 2001).It also improves the economy of water and carbon, maximizing growth and even strengthening transplanting.In garlic, the use of vermicompost produces an earlier start of bulbi cation and increases the assimilate partitioning index (Argüello et al., 2006).
Vermicompost application to lettuce seedlings signi cantly improved growth (Atiyeh et al., 2002), increased lettuce both fresh and dry biomass weights (Ali et al., 2007), and the enhanced plant weights were independent of the nutrient content of the substrates.In other crops, this biofertilizer greatly increased foliar area and biomass (Lima & Silva, 1998).Treatments involving vermicompost generally stimulate growth.Atiyeh et al. (2002) also mention a number of references in the literature showing that plant growth regulators, such as auxins, gibberellins and cytokinins, are produced by microorganisms.It has been suggested that the promotion of microbial activity in organic matter by earthworms may result in the production of signi cant quantities of plant growth regulators (Krishnamoorthy & Vajranabhiah, 1986;Tomati et al., 1983Tomati et al., , 1988Tomati et al., , 1990;;Tomati & Galli, 1995;Edwards, 1998).Earthworm activity accelerates the humi cation of organic matter, and its in uence in increasing microbial populations enhances the presence of auxins and gibberellin-like substances as well as humic acids (Casenave de San lippo et al., 1990).Similar results were presented in biosolids by Zhang et al. (2009).
From an anatomical point of view, it has been determined that the primary growth of plants has its origin at the level of the ground meristem, procambium and protoderm (Dickinson, 2000;Evert, 2008).Mineral nutrition contributes to structural organization, since anatomical modi cations are found when plants receive fertilizers, which can alter tissue thickness (Marschner, 1995).Anatomical studies in co ee to determine the e ects of nutrients on anatomy have shown that modi cations induced in tissues can also in uence assimilate partitioning (Rosolem & Leitte, 2007).However, there is no information in the literature to date that explain how vermicompost a ects the anatomy and physiology of lettuce seedlings, and impacts assimilate partitioning (Marschner, 1995).
e xylem is the tissue that transports water and minerals from the root system to the aerial portions of the plant, and the phloem translocates the products of photosynthesis from mature leaves to areas of growth and storage, including the roots.e photoassimilates move from the production zones, called sources, to metabolism or storage zones, called sinks.A mature leaf is capable of producing photosynthates in excess of its own needs (Taiz & Zeiger, 1998) Our hypothesis is that vermicompost stimulates lettuce plantlet growth acting at the ground meristem and procambium levels of the shoot, and also optimizes physiological aspects, such as biomass increase and assimilate partitioning.
e aim of this study was to analyze the impact of vermicompost on lettuce seedlings as regards anatomo-physiological modi cations related to assimilate partitioning and growth.

MATERIALS AND METHODS
Plant material.Young lettuce (Lactuca sativa L.) var.Criolla Verde plants were grown under greenhouse conditions at the Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina.e greenhouse conditions were: temperature between 20 and 25 °C, relative humidity of approximately 70% (± 20%), and natural light.e growth medium was soil (Entic haplustoll) with and without vermicompost taken from solid waste from industry fridge (All Green Company).
Treatments were (a) soil control (C) and (b) 1 soil: 1 vermicompost (by volume) (V).e physicochemical parameters evaluated were organic carbon, phosphorus, total nitrogen, electrical conductivity and pH.e organic C content was determined by Walkley-Black (Nelson and Sommers, 1996).Total N was evaluated by Kjeldahl and extractable P by Bray and Kurtz Nº1 (Kuo, 1996).e pH values were measured in aqueous extracts 1:2.5 with a pH meter (Orion Research 901).Electrical conductivity was measured in saturated paste with a conductivity meter (DIST4 of HANNA Instrumental).

DISCUSSION
Previous studies demonstrated that lettuce seedlings treated with vermicompost showed greater vigour and growth (Ledesma et al., 2001); the experiments reported here gave similar results.One possible explanation for this is that a signi cant increase in aerial biomass may be generated with a consequent increase in photosynthesis (Fig. 1 A).Other authors have also found increases in biomass and foliar area in lettuce (Sganzerla, 1983;Mujahid & Gupta, 2010), in tomatoes (Atiyeh et al., 2002) and coriander (Lima & Silva, 1998).
is is explained in terms of the increase in NAR (Fig. 1 B), which is related to a lower FAC in the treatment with vermicompost, indicating greater e ciency in the production of photoassimilates (Fig. 2 A). e present investigation demostrated that this increase in NAR is due to an increase of approximately 50% in the number of cell layers and in mesophyll height (Fig. 3: A-3, B-3 and Fig. 4). is increase can most probably be explained by the e ect of vermicompost on the number of mesophyll layers, increasing the NAR.
In this context, it is worth asking what plants treated with vermicompost do with their greater production of photoassimilates.Previous ndings indicate that vermicompost changes the assimilate partitioning pattern, prioritizing distribution to the aerial part in lettuce (Ledesma et al., 2001), and to the bulb in garlic (Argüello et al., 2006).
How then can this greater distribution be explained from the anatomical point of view?e results suggest that vermicompost acts to increase the number of vessel members (Fig. 3: B-1 and Fig. 4 C) and the phloem area (Fig. 4 D), and it also seems clear from the above discussion that vermicompost increases procambium activity.ese anatomical modi cations are consistent with a greater e ciency in the assimilate partitioning, which is seen in a decrease in the FAC and SLA Coe cients (Fig. 2 A-B), which in turn, accounts for the greater Harvest Index (Fig. 2 C).
A deeper analysis of Figure 4 (A-B) reveals that the ground meristem activity is manifested in the vermicompost treatment by an increase (1) in the number of mesophyll layers at 30 days after sowing, and (2) of the mesophyll thickness at 45 days after sowing.On the other hand, it is important to point out the increase in the number of vessel elements (Fig. 3A and 4C) and phloem area (Fig. 3B and  4D) which occured after 15 days from sowing, as a result of the same treatment.is can be linked to anticipation in the activity of the procambium.ese might be explained by the presence of growth hormones in the vermicompost, wich are absorbed in humic acids, forming complexes (Atiyeh et al., 2000(Atiyeh et al., , 2002;;Arancon et al., 2004Arancon et al., , 2006;;Edwards et al;2006).e hormones present constitute an external "signal" that acts on the primary meristems mentioned earlier, which are responsible for the perception of signals that stimulate growth (Argüello et al., 2008).
In conclusion, experimental evidence suggest that the vermicompost increases growth showing a greater generation and translocation of photoassimilates, which can be explained in terms of various anatomical modi cations found.