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Sunday, August 23, 2026

Anthropology, Participatory Research and Development Cooperation: A Necessary Dialogue- Juniper Publishers

Archaeology & Anthropology- Juniper Publishers

Abstract

Le domande sul “perché e per chi” if it generates conoscenza [1] sono da tempo a chiave per anthropological discipline. At the present time, it is always the case that it is important for the dissemination of the risultati, poiché il sapere prodotto in such a way that it can be used positively in the realta study. This line of thought is directed towards the end of the rifle and the gli obiettivi of the progetti of cooperation to the sviluppo, in particolare i micro-progetti. The text presents some concrete cases in which, as an anthropologist, I directed cooperation projects and community formation processes in indigenous areas of Mexico. These experiences reveal the need for integrating anthropological research and cooperation activities, in which the researcher’s work has facilitated the identification of key issues for the intervention and the relationship with the community that hosted it. Secondly, the necessary complementarity of knowledge for the integral interpretation of reality and the consequent design of cooperation projects became evident. Thirdly, the enhancement of local organizational structures guarantees the strength and durability of the results obtained through cooperation activities.

Keywords: Participatory research; Development cooperation; Anthropology; Mexico

Introduction

“We do not ask for your help, rather we want you to come and share our cause and our struggle.” This is what Juan told me, representing a Zapatista indigenous village in Chiapas, in southern Mexico, during my first research trip for the realization of my thesis in Ethnology. An irrefutable ethical statement that should accompany every research or cooperation project, both too often marked by welfarist or ethnocentric legacies

At the age of twenty, I found a way to reach the south-east of Mexico by participating in a project of “cooperation from below” directed by a small Italian NGO: for a month, I found myself with other volunteers doing masonry work for the construction of a turbine that equipped the village with electricity. In the twenty years that followed, anthropological research turned into my life project, always intertwined with projects of cooperation and community formation in indigenous areas of Mexico. Juan’s words have turned into the ethical compass that, both in research and in cooperation, has always sought the joint construction of projects and results with the stakeholders. The experiences realized also reveal the need for the integration of anthropological research and cooperation activities, in which the researcher’s work facilitated the identification of key issues for the intervention and the relationship with the community that hosted it.

Discussion

Anthropology and cooperation: from coloniality to participation

First of all, I stress the obvious parallelism between anthropology, as a social science, and development cooperation, which have in common a colonial or neo-colonial root. The anthropological discipline developed, historically, with the declared objective of producing knowledge on the Others, indigenous and native populations, object of the colonial domination. Also, in Mexico, the exceptional development of the discipline since the thirties is part of the same process, expression of the nationalist policies of state strengthening, assimilation and control of cultural diversity, defined by González Casanova [2]as internal colonialism.

Secondly, “development cooperation” also bears the colonial mark defined by the controversial term “development”. The criticisms of the concept are manifold; among the many authors, I would like to mention here only [3-5] which from distinctperspectives have shown how development implies the action of transforming subjects and directing them to change towards an objective defined elsewhere: “progress”, often to the detriment of the way of life, the relationship with the environment and the cosmovision of the “subjects / objects” of development itself. The limits and potential of cooperation have long been highlighted by the current anthropology of development or anthropology of development [6-9].

Starting from the identification of the common colonial and neo-colonial brand, the reflections on the relevance and purpose of the research or of the cooperative intervention are also common; on the recipients and producers of the knowledge produced or shared, on the ways in which these are produced. Finally, the question concerning the “why and for whom” social sciences create knowledge, formulated by Robert Lynd since 1939, continues to be central to the anthropological discipline. The construction of knowledge, infrastructure or training are processes that have in common the objective of transforming existing reality, and as such require the active participation of both the local population as well as researchers and cooperators. If anthropology is understood as a political practice, and therefore with a relevant theoretical substrate, a complex ethnography and a political commitment, it can therefore have a real impact in cooperation policies and projects: it would be the passage from an anthropology of development to a postdevelopment anthropology [10].

With the intention of subverting the neocolonial heritage, research approaches have been developed since the 1960s that favor the participation of those who previously were considered only “objects of study” and, subsequently, objects of the intervention of public policies and processes of building ethnically hierarchical societies. It is in this age that, in different countries, the enhancement of “local” or “popular” knowledge is growing and the proposal of participatory action research (IAP) promoted by Fals-Borda in the Seventies is born. The centrality of research as a tool for the transformation of society also characterizes the approaches marked by the reflection on the intrinsic colonial brand or coloniality of power and knowledge [11], or those focused on methodological reflection, such as activist or militant anthropology [12]. With increasing frequency, research projects are developed in which the “subjects of study” actively participate, from the definition of the themes or co-licensing [13] to the dissemination of the results, since the knowledge produced in this way can be used for affect positively in the studied reality.

Participatory action research is at the same time a research methodology and a social transformation project, since it involves the active participation of the population, which goes from being an object to the subject of research aimed at social change [14]. The question of Lynd on why, for whom and how to do research is equally fitting if transposed to the theme of international cooperation. The emphasis on participation in all phases of social transformation is key in the decolonization process, both in anthropology and in cooperation. Both pass both for a democratization of projects and activities (research and development cooperation), which in order to acquire cognitive and social effectiveness and relevance must be forcibly shared and consensual with the subjects. The active participation of the collective subjects involved in the project is fundamental, from the identification of the problem, to its analysis (object of the participatory research) up to the elaboration of the intervention project, its planning and the realization of the activities (objective of the projects of cooperation). In this sense, there is a necessary relationship of continuity between anthropological research and participatory and bottom-up cooperation projects [9] a field of action studied and theorized by the current anthropology for development [15] or development anthropology [7].

Anthropology and cooperation: an experience from the field

The experience of one of the various cooperation projects in indigenous regions of Mexico in which I participated allows me to exemplify some further reflections. This is the Community Communication Project promoted by the Coordinadora Regional de Autoridades Comunitarias, in the Costa Chica and Montaña regions of the state of Guerrero. Between 2005 and 2014 I carried out research activities in the area, and in particular on the activities of the organization mentioned, which brings together different indigenous peoples. The relationship of trust built with the organization and with the inhabitants of the villages, thanks to a method of participatory research, meant that I was shared a need that had been identified for some time: the impulse to one’s own, direct means of communication to facilitate the exchange of information both within the region and externally [16].The collective discussion led to the definition of two main instruments: a network of community radios that spoke the indigenous languages of the region and gave space to local problems, and a web page that spread the activities of the Coordinadora Regional de Autoridades Comunitarias.

The first requested contribution was in the participated elaboration of the project, and subsequently in the funding research. Cooperation agencies outside the region did not intervene in this case, but in a collaborative way (through assemblies in which the region’s indigenous authorities participated, and then village by village) a shared project was drawn up, and then they looked for the sources of financing. In this case, considering the micro character of the project, we turned to funding sources that did not require a high level of formality in the project (The German Umverteilen foundation; the cooperation sector of the Italian Region Trentino-Alto Adige through the mediation of an Italian NGO). In this context, the voluntary cooperation of the villages concerned with materials for the construction of radio studios, labor or money was fundamental. My role was, after the identification of the need and the participation in the elaboration of the project, substantially that of mediator between the indigenous organization and the requests for funding, and the coordination of the activities that required the presence of subjects external to the region (training of young speakers and editors, installation of radios).

The role of the anthropologist can therefore provide different contributions to the world of cooperation. First of all, from the anthropological research work needs can emerge which are then channeled to the development of cooperation projects, favoring a real relevance of the projects. Secondly, anthropological knowledge, which consists of a profound knowledge of the cultures on which one acts, can provide a multiplicity of visions that guide cooperation and help it to define its interventions in a culturally appropriate way. According to Antonino Colajanni, an anthropologist who has worked on these issues for decades, the first thing to check for a future project is its “socio-cultural sustainability” that can be ascertained with a survey on local capacities to generate resources, with skills, knowledge, interests and vision of the future. The best idea is to ensure that the next intervention does not create dependence but stimulates local capacities [17]. An anthropologist does this by profession: he observes, questions, tries to understand, listens, feels, sees, understands the links between people and has a technical knowledge that allows him to collect this “forgotten dimension of development”, the cultural one

Complementarity of knowledge and enhancement of own knowledge

Collaborative anthropology, and truly such cooperation, have in common the enhancement of local skills and knowledge. In training activities on the subjects of popular communication, community journalism and radio communication aimed at the young operators and operators of indigenous radios installed in Guerrero, we have indeed invited external experts, but we have also and above all involved those subjects present in the territory (masters, lawyers, but also seniors, students and peasants) whose skills have been valued within the training activities. In this way the territorial rooting of the project is strengthened, and the knowledge that is conveyed is culturally appropriate; there is no need for a “translation” of the cooperation objectives because the project is acted by the subjects themselves.”Solo entre todos sabemos todo”, explained a wixárica indigenous essay. The phrase highlights the necessary complementarity of local, cultured, popular, traditional and innovative knowledge for the integral interpretation of reality and the consequent design of cooperation projects. We are in the sphere defined by Sousa Santos as “ecology of knowledge”, where “the credibility of a cognitive construction is measured by the type of intervention in the way it allows or prevents” [18].

Enhancement of local organizational structures

Following the discussion of the Community Communication Project in the assemblies inside each village, these collectively chose the people who would participate in the training activities (mostly young people) and consequently they would then be in charge of the community radio operation. In this way the project was legitimized and appropriated within the local society and culture. The leaders of each of the three radios were then elected in the Regional Assembly (attended by all the representatives of the villages in the region). In this way that of “radio manager” becomes a public office, recognized and included in the traditional indigenous organizational structure; this guaranteed the durability of the results, since the vigilance on the good technical and organizational functioning of the radios was immediately assumed by the local communities.

Conclusion

The experience I told is of micro-cooperation, and obviously the cooperation on a larger scale, which implies a higher technological specialization and greater economic investment, faces more complex problems. In any case, I believe that the emphasis on sharing, participation and the substantial democratization of cooperation, as well as the production of knowledge that should accompany it, must be key elements to be privileged in activities at every scale, micro and macro. The anthropological sensitivity, attentive to cultural nuances and to complex social transformations, is also characterized by the continuous movement on the micro and macro scales of knowledge, and in this, I believe, its fundamental contribution to the world of cooperation should be enhanced and strengthened.

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Thursday, August 20, 2026

The “State of The Art” of Art Therapy in Psychiatry: Reflections on International Evidences and Italian Experiences- Juniper Publishers

Complementary Medicine & Alternative Healthcare- Juniper Publishers

Abstract

The complex and reciprocal dialectic between art and psychiatry has represented the object of investigation of many researchers for many years in last century, although a specific role of art therapy in psychiatric. The art-therapies are currently conceptualized as forms of psychotherapy employing artistic media (painting, dance, theatre, music etc.) as an expressive and receptive communication modalities. In Italy, along the XX century there has been a growing diffusion of art-therapies as rehabilitation techniques in several settings. The currently available empirical evidence suggests a relevant role for art-therapies in psychiatry, especially for psychotic’s patients with negative symptoms and depression. This brief summary has not set as its objective a critical reevaluation of all the studies present in the literature on Art Therapy, a task left to the experts of the meta analyses and reviews but wants to be a reflection on some limits of the current studies. Final point is on the Italian reality: despite the evidence and the ten-year tradition on Art Therapies in Italy, they have a fairly wide spread in the rehabilitation services of the Mental Health Departments and Art Therapy is still confused as “socialization” or as techniques of which little is known about the effect. This confusion refers to the need for training on the subject to be constant for psychiatric service operators and for scientific researchers to produce evidence that can be used to finalize its use in a personalized way with a view to an increasingly modern precision medicine.
Keywords: Art therapy; Psychosis; Depression; Psychiatric rehabilitation

Introduction

The complex and reciprocal dialectic between art and psychiatry has represented the object of investigation of many researchers for many years in last century [1-9]. In the last few decades the concept of “Art Therapy” has been developed and reshaped in many different ways and has often been associated with various Anglo-Saxon expressions such as “Creative Art Therapies” or “Expressive Therapies”. Following the definition given by the British Association of Art Therapists, Art Therapy is a “form of psychotherapy that uses graphic-pictorial artistic means, but not only, as a primary mode of communication” [10]. This definition, however, favours a conceptualisation of Art Therapy meant as “art in therapy” (art represents an expressive and emotionally immediate means to be placed within a more structured psychotherapeutic path), to the detriment of the conceptualization known as “art as therapy” (that is, art as a form of re-socializing therapy and that eases emotional expression itself), doubtful for many, but still widely represented in the context of psychiatric rehabilitation [11,12].
In Italy, Art Therapy has experienced a substantially bimodal spread. An initial interest in the use of the arts in psychiatry in Italy lies at the end of world conflicts and has substantially followed, in the context of psychiatric hospitals, the international orientation ruled by the current psychoanalytic thought [13,14]. In the subsequent decades of the twentieth century, different art-therapeutic approaches have spread in Italy, based on empirical evidence (although not systematically collected and sometimes anything but methodologically flawless) relating to the good impact on resocialisation processes and reasonable costs. However, after the gradual closure of psychiatric hospitals, the parallel shift towards community-oriented psychiatry paradigms, the critical review of psychoanalytic paradigms, associated with an indiscriminate (or, more often, inappropriate) application of the above mentioned techniques have probably led to a drift that has dragged the Art Therapies, like other rehabilitative approaches, increasingly towards entertainment and less and less towards a real psychiatric rehabilitation [15-18].
With the transition to the third millennium, the use of art in psychiatric rehabilitation has experienced a new impulse associated with greater methodological rigor and the accumulation of growing scientific evidence. Several randomized controlled trials have been conducted and they have demonstrated the effectiveness of such rehabilitative approaches as an additional treatment in different mental and neurological disorders [19-29]. This second peak of interest in the application of artistic disciplines to psychiatric rehabilitation was not only associated with a greater methodological rigor in documenting the effectiveness of these approaches, with standardized and reproducible outcome assessments, but also and above all through the application of reference models that are no longer exclusively psychoanalytic (many approaches of cognitive, narrative, systemic-relational orientation, centered on the person, etc. have spread), extension to populations (not just patients with chronic psychoses, but patients with various psychiatric diagnoses, often with a recent history of illness and different levels of severity) and different settings (above all, territorial psychiatric facilities and rehabilitation residences, in short-term and long-term programs) as analyzed in recent reviews and meta-analysis [30,31]. Finally, the National Collaborating Center for Mental Health [32] has also included the “Art Therapies” among the recommended treatment to promote (together with a structured psychotherapy) the healing of patients with schizophrenia, especially if young and with prevalent affective and/or negative symptoms. In the last decade some evidences of its effectiveness on specific psychological and symptomatological dimensions and in different age groups are appearing, even if an agreement does not yet exist.

Art Therapy and Psychotic Disorders

A few years have passed since the well-known RCT of the English group of Mike Crawford (Matisse Project Team) [33] which, through a severe RCT, stated that the use of Art Therapy was not more effective than other standard techniques in improving global functioning for schizophrenic patients [34]. Since then, studies have appeared in literature that, always analyzing the therapeutic effect of Art Therapy for schizophrenic patients, have used less quantitative standard dimensions as indicators, that show that they seem to reduce the expression of negative symptoms in schizophrenia, improve self-knowledge, interpersonal skills and affective development. For example, another study always conducted in the United Kingdom on acute psychiatric inpatients has stressed that participation in structured sessions of Art Therapy can change the subjective experience in a positive sense and restore a sense of hope in the future, increase participants’ level of emotional awareness and ability to reflect on other people’s emotional states, thus indirectly acting on positive affective dimensions and recovery [35]. Very similar is the result of another study conducted in Norway [36] that aimed more at investigating the mechanisms of action of Art Therapy rather than the effects on psychiatric symptoms, observing an improvement in the insight and management of positive symptoms. The authors confirm that Art Therapy is effective especially when inserted into integrated treatment programs.
In a meta-analysis conducted by De Silva MJ et al. [37] the Art Therapies still show common positive efficacy profiles like other psychosocial interventions conducted for psychotic patients.
Angelica Attard and Michael Larkin published a review of the literature on the effectiveness of Art Therapy for psychotic patients in 2016 and concluded that valid studies are still a small number in literature. Among these 18 High-quality quantitative articles provided inconclusive evidence for the efficacy of Art Therapy in adults with psychosis. However, high-quality qualitative articles should be considered as Art Therapy to be beneficial, meaningful, and acceptable, although this conclusion is based on a small number of studies [38]. A limitation inherent in randomized clinical trials that aim at measuring the effectiveness of Art Therapy in psychotic patients is the use of standard symptom outcome indicators (positive symptoms, negative symptoms) [39] while it would be more appropriate to use more extensive indicators that also take into account qualitative and subjective dimensions and emotional experiences, indicators more suitable to measure the subjective effect of these techniques that have a psychotherapeutic value. In a study conducted on patients dwelling in community facilities (psychiatric rehabilitation community and day center) using the ESM (optimal experience or flow method, to evaluate a state characterized by the perception of high challenges and high skills, deep concentration, positive affect, clear goals, control and autonomous motivation, which contributes to individuals’ well-being) this state is most evident in some rehabilitation activities including Art Therapy and instead does not occur in non-structured or leisure time [40]. Several authors have emphasized as a limit in the design of RCT for these techniques the need for patient motivation to the activity, a condition that enables a constant adherence to the activity itself [34].

Art Therapy and Non-Psychotic Disorders

A recent 2017 review of the Uttley and Sutton (UK) group conducted on articles published up to 2013 highlighted that benefits associated with Art Therapy included the following: the development of relationships with the therapist and other group members; understanding the self/own illness/the future; gaining perspective; distraction; personal achievement; expression; relaxation; and empowerment. Small numbers of patients reported varying reasons for not wanting to take part, and some highlighted potentially negative effects of Art Therapy which included the evoking of feelings which could not be resolved. The findings suggest that for the majority of respondents Art Therapy was an acceptable intervention, although this was not the case for all respondents. Therefore, attention should be focused on both identifying those who are most likely to benefit from Art Therapy and ensuring any potential harms are minimized [41].

Art Therapy and Action on Internalizing Symptoms and Depression

Most studies in the literature on art and depression concern the elderly population. Dunphy K et al. [21] have published a systematic review on the use of artistic techniques for elderly depressed patients, selecting those conducted by specialized professionals came out to confirm the use of artistic techniques in the improvement not only of mood and of physical state of the people, more connected to the techniques like dance movement- therapy and dramatherapy but more generally of the subjective perception of well-being and enhanced self-concept, of the processes of elaboration of the emotions, of the cognitive functions like memory [20,21].
Less data are available on the very young even if for the 14- 18 age group, in a controlled clinical study, we have seen that participation in Art Therapy sessions for short periods (6 sessions) improves the dimension of internalization, which is often linked to depressive and anxiety symptoms and withdrawal, thus providing a more solid theoretical basis for the use of these techniques in adolescents with anxiety, depression and withdrawal disorders [42].

Conclusion

This brief summary has not set as its objective a critical re-evaluation of all the studies present in the literature on Art Therapy, a task left to the experts of the meta-analyses and reviews, but wants to be a reflection on some limits of the current studies that use only the impact of the technique on symptom resolution as indicators, classic approach for clinical trials on psychiatric drugs or punctual interventional techniques (surgery) but less suitable to probe the effect of intervention techniques that act on more complex dimensions of the person, such as self-perception and of one’s emotions, the ability to get in touch with one’s own intrapsychic experience, with the emotions of others, without falling into the classic bias of product measurement (artistic product) because the product of the creative action of the Art Therapy session in psychiatric rehabilitation often finds its explanation in the dimension of the relationship more than in the product itself. What is now shared by several authors is that Art Therapy is not improvisation, it is a psychotherapeutic and rehabilitative technique that requires specialized training to be applied; it is therefore a technique that cannot be improvised.
Despite the need for trained personnel, it seems that in the studies on the costs of the different interventions, Art Therapy does not seem more expensive than other ones, as highlighted by Lesley Uttley in 2015 with the PROSPERO study for the National Institute for Health Research Health Technology Assessment [29] where he states that Art Therapy was associated with positive effects when compared with a control in a number of studies in patients with different clinical profiles, and it was reported to be an acceptable treatment and was associated with a number of benefits. Art Therapy appeared to be cost-effective compared with waitlist, but further studies are needed to confirm this finding as well as evidence to inform future cost-effective analyses of Art Therapy versus other treatments. Final point on the Italian reality. Despite the evidence and the ten-year tradition on Art Therapies in Italy, they are still perceived as “socialization” or as techniques of which little is known about the effect. Although expressive techniques, including Art Therapy, have a fairly wide spread in the rehabilitation services of the Mental Health Departments, reaching about 10.5% of all structured group activities implemented in Lombard rehabilitation services [43] and about 8.5% in the national average, as shown in a survey conducted in 2013 on the national territory by the Italian Society of Psychosocial Rehabilitation [44]. There is still a lot of confusion among operators in differential terms between what is defined as an artistic expressive technique and what is instead a simple socialization intervention or an occupational activity during which sonorous means are used, such as singing, drawing, or still production of artefacts. These interventions also have their own dignity but cannot be defined as “expressive” and are often conducted by operators without specific training in arts-therapies or at least in psychotherapy. In the reality of psychiatric services it is not rare to see activities such as singing, painting, decoration, production of artefacts or to generally socializing activities (for example, karaoke) conducted by personnel without a specific artistic or psychotherapeutic training defined as “Art Therapies” or structured group activities led by trained therapists to be ascribed into the performance registration system as “group socializing activities”. This confusion refers to the need for training on the subject to be constant for psychiatric service operators, taking into account the turnover that is occurring in this field and for scientific research to produce evidence that can be used to finalize its use in a personalized way with a view to an increasingly modern precision medicine.
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Wednesday, August 12, 2026

Suppression of Bean Yellow Mosaic Virus by Plant Extracts in Faba Bean Plants- Juniper Publishers

Organic & Medicinal Chemistry- Juniper Publishers

Abstract

The virus was obtained from faba bean plants that were naturally infected. The studied virus was identified as Bean yellow mosaic virus (BYMV) according to symptomology, host range, virus stability, modes of transmission, ELISA, and RT-PCR. ELISA test confirmed the presence of BYMV in all the collected leaf samples at different times after inoculation. A fragment of coat protein gene (352 bp) was amplified using PCR from infected faba bean plants. Growth and seed characters were significantly lower in infected plants compared with healthy faba bean plants. Plant extracts promoted systemic resistance to BYMV. Disease severity and BYMV titre were substantially decreased in faba bean plants treated with extracts of Cinnamomum zeylanicum bark (CZ), Syzygium aromaticum, Matericaria chamomilla L., Foeniculum vulgare, Zingiber officinale before or after BYMV inoculation. The lowest disease severity and virus concentration were achieved using cinnamon extracts treated at 1 day before or after virus inoculation. In faba bean plants treated with plant extracts, the expression of pathogenesis-related genes (PR1 and PR2) was substantially higher than in control plants. These results explain the role of PR1 and PR2 genes in enhanced resistance against BYMV by plant extracts.

Keywords: Faba bean; Bean yellow mosaic virus; Induced resistance; ELISA; Real time PCR

Introduction

In Egypt, faba bean (Vicia faba L., Fabaceae) is a main food legume crop. It represents an important source of protein, carbohydrate, vitamins, essential amino acids, and mineral salts [1]. It is used as a human food in developing countries and as animal feed in industrialized ones, mostly for pigs, horses, poultry, and pigeons. It may be eaten raw or cooked, fresh, or preserved. After infecting the susceptible host plant, viruses cause a sequence of physiological changes that may lead to disease symptoms such as systemic and local symptoms [2,3]. BYMV (Bean Yellow Mosaic Potyvirus) is a widespread disease of beans and other hosts that may be found all over the globe [4]. It is a severe disease wherever susceptible crops grown because it is transmitted through seeds and Aphids [5]. Recently, Skelton et al. [6] isolated and identified BYMV from Dactylorhiza foliosa (a hardy orchid species, native to the Island of Madeira) that showed symptoms of chlorotic mottle and streaking. Finally, the virus was mechanically inoculated into two indicator species. Chenopodium quinoa (chlorotic local lesions) and Nicotiana benthamiana (distortion and mosaic) developed leaf symptoms. The presence of BYMV was verified by ELISA testing of plants exhibiting symptoms.

Several investigators found that faba bean was the most susceptible host to BYMV causing the considered losses in the grain yield [7]. This virus was isolated, either alone or mixed with other viruses, from faba bean. Systemic symptoms produced by BYMV infection may not kill faba bean plants, but they can spread quicker and deeper throughout the crop, causing a larger total yield loss, despite inducing milder symptoms [8,9]. A long-known virus inhibitor from carnation leaves has now been shown to be an inducer of systemic resistance to virus as well [10]. Thus, it is possible that many of the other well-known virus inhibitors of plant origin may in fact be proven to be inducers of systemic resistance. The earliest evidence that plants possess inhibitory compounds came from virus-infected plants, and then viral inhibitory substances were discovered in a variety of healthy plants from several Angiosperm families, including Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Solanaceae, and Verbenaceae [11].

While working on resistance induced by leaf extracts from carnation plants, they observed that a very short period was required to the induced antiviral state in host tissue [10]. Pyrethrin (Pyrethrum) is a source of synthetic pyrethroid insecticides that is generated in the flowers of Chrysanthemum cinerariaefolium. Pyrethrin is approved for the use against a wide range of pests. One formulation of pyrethrin was shown to be moderately to very efficient (61-100 % control) against the fruit pests, such as grape leafhoppers, potato leafhoppers, leaf curl plum aphids, blueberry flea beetles, blueberry thrips, and blueberry sawfly. It also works against the cranberry fruit worm. It decomposes rapidly in the environment and may be used up to harvest day [12].

Ribosome-inactivating proteins (RIPs), which have antiviral effects, are found in many plant species [13]. A RIP from pokeweed (Phytolacca americana) was shown to provide broad-spectrum viral resistance in tobacco and potato. All the tested RIPs exhibited strong antiviral action [14]. Purified antiviral proteins from roots, shoots, leaves, fruits, and seeds of Mirabilis jalapa were successful in protecting economically significant crops (tobacco, maize, and potatoes) against a wide range of plant viruses such as Tobacco mosaic virus and Tomato spotted wilt virus [15,16]. This study aims to isolate of the most common faba bean virus found in collected samples and to evaluate the induced systemic resistance activities of some medicinal plant extracts against Bean yellow mosaic virus.

Materials and Methods

Isolation and identification of the isolated virus

One type of naturally infected faba bean samples showing virus-like symptoms was collected from faba bean fields, Legume Department, Sakha Agricultural Research Station, Kafr EL-Sheikh Governorate. According to Elsharkawy et al. [17], mechanical inoculation was employed to inoculate the test plants (faba bean). The viral isolate was biologically purified using Chenopodium amaranticolor L. and the local lesion method was used [18]. The Sakha Agricultural Research Station in the Kafr El-Sheikh Governorate, Egypt, provided sixty plant species and cultivars from ten distinct families that were mechanically inoculated by the isolated virus to study the host range.

Isolation and identification of the isolated virus

Serological diagnosis of the isolated virus using indirect ELISA

Faba bean samples showing symptoms suggested to be due to Bean yellow mosaic virus (BYMV) was tested with their specific antiserum by indirect ELISA technique. This method was described by Elsharkawy et al. [19]. Leaves of faba bean were ground in a mortar with 0.02 M phosphate buffer (1:10) and mix the sap 1:1 with 2x concentrated coating buffer, pH 9.6, fill the wells with 200μ1 aliquots of test samples. The plates were covered and incubated at 30- 37 °C for 2 hrs. Washing with PBS-Tween was repeated 3 times. Cross-absorbed antiserum (200 μ1) was added to each well then Incubated for 1-1.5h at 30-37°C followed by washing with PBS-Tween. Goat anti-rabbit antibody conjugated to alkaline phosphatase (200 μ1) was added and incubated for one hour at 37°C followed by washing with PBS-Tween. P-nitrophenyle phosphate (200 μ1) was added and the absorbance values was measured at 405nm using Vniskan ELISA reader.

Extraction of total nucleic acid from plant tissues infected with BYMV

The Spin/ vacuum SV total RNA isolation system (Promega Corporation, Maison, WI) has been used successfully to isolate RNA from leaves of healthy or BYMV infected faba bean plants. Thirty mg of infected and uninfected faba bean leaves were powdered in liquid nitrogen. One hundred seventy-five μl of RNA lysis buffer (4M Guanidine thiocyanate, 0.01 Tris-HCl, pH 7.5, 0.97% β- mercaptoethanol) was added and the tube was mixed by inversion. The dilution buffer was added, and the tube was mixed by inversion followed by centrifugation at 15000rpm in a microcentrifuge for 15minutes. RNA precipitation and DNase treatment were carried out as described by Elsharkawy et al. [17]. RT-PCR primers used in this study were designed to detect the coat protein gene of BYMV as described by Shooman [20].

The selection of the primers was performed according to the primer analysis software Oligo 4.1 (National Bioscience Inc., Plymounth MN, USA). The primer bought from integrated DNA Technologies, Inc. The primer pair used for the detection of BYMV (NIF-5’-GAGCGCATCGTTTCAATTCT-3’ and NIR-5’- AGCATGGGGCTATCCAACT-3’) was designed based on GenBank accession AM884180. The PCR primers utilized for cDNA synthesis and amplification of the BYMV coat protein gene were based on the conserved sequence of other BYMV isolates from all over the world. The purified total RNAs extracted from infected and healthy faba bean plants by using SV total RNA isolation system were used as starting material in the Reverse transcriptase polymerase chain reaction (RT-PCR) process as described by Shooman [20] and Hataya et al. [21]. the PCR products were examined on a 1% agarose gel electrophoresis and the size of the full length BYMV DNA fragment was determined in accordance with the DNA molecular weight markers.

Induction of systemic resistance against BYMV by plant extracts

Extracted oils from five medicinal plants (Cinnamomum zeylanicum withbark (Cinnamon), Syzygium aromaticum (Clove), Matericaria chamomilla L. (Chamomile), Foeniculum vulgare (Fennel), Zingiber officinale (Ginger) were kindly provided by Horticulture Department, Faculty of Agriculture, Kafrelsheikh University. Plant extracts (oils) were stored in the refrigerator at 4°C until use. The isolated virus inoculum was prepared by triturating 1 g of young viral symptomatic faba leaves cv. “Giza 843” in a sterilized mortar by adding 20ml (1:20 w/v) of sterile distilled water. The extract was filtered through muslin cloth then centrifuged for 20min at 3000rpm. The supernatant was used as inoculum.

Plastic pots (25cm) were filled with suitable amount of sandclay- loamy sterilized soil mixture. Ten seeds per pot were planted and subsequently trimmed to three seedlings after 15 days from planting. When needed, pots were irrigated with tap water in equal quantities. NPK fertilizers were used (0.6g of urea/pot, 0.75g of Ca-super-phosphate/pot, and 0.25g of K-sulphate/pot). Phosphorus was supplied before sowing during soil preparation. N and K were supplied in two equal dosages at thinning and two weeks afterwards.

Faba bean plants were divided into eleven groups (15 plants for each in 5 pots), five of them sprayed 24 hours pretreatment (virus-inoculation) with medicinal plants. Five groups were sprayed 24 hours post BYMV-inoculation with medicinal plants. The last group (15 plants) served as control, pretreated with water, and subsequently inoculated with the sap from virus-infected plants. Crude extracts of selected medicinal plants adjusted as 1:5 w/v and sprayed using pressure sprayer (2L). After two weeks from virus inoculation, virus concentration was determine using indirect ELISA as described previously and also disease severity of BYMV was measured based on the following scale: 0=no symptoms; 2=mild mosaic of the youngest two leaves; 4 = pronounced leaf deformation and mosaic of the youngest two leaves; 6=pronounced leaf deformation and mosaic with progression of symptoms into sequentially older leaves; 8=pronounced leaf deformation and mosaic, with all leaves expressing some forms of BYMV induced symptoms and 10=similar symptoms as described for a rating of 8, with plants also being stunted in growth.

Molecular investigation of pathogenesis-related genes expression

The kit (Thermo Scientific, Fermentas, #K0731) was used for RNA extraction. Pure RNA was reverse transcribed using the reverse transcription kits (Thermo Scientific, Fermentas, #EP0451). RNA was quantified using Nanodrop. The Q5000 (Uv- Vis’s spectrophotometer Q5000/USA) automatically performs all necessary measurements and calculations. For pure RNA, the OD260/OD280 ratio is less than 2. Protein contamination (which has a maximum absorbance of 280 nm) or phenol contamination will result in a ratio that is considerably lower than these values. The expression of target genes (PR1 and PR2) was assessed using real-time PCR with SYBR Green, with elongation factor 1 alpha (EF1α) as an internal reference following the manufacturer protocol (Thermo scientific, USA, # K0221) and gene specific primers. Table 1 lists the primers used in the amplification. The housekeeping gene (EF1α) is utilized to determine the relative gene expression or fold change in the target gene. Therefore, the 2-ΔΔCt technique was used to normalize the amounts of the target gene’s critical threshold (Ct) with the quantities of the housekeeping gene (EF1α) [22].

Statistical Analysis

The data were all presented as means (±S.E.). Comparisons were obtained using Duncan’s multiple range test (DMRT) and statistical significance was determined using one-way analysis of variance (ANOVA) by SPSS 18.0 software, 2011. When p<0.05, values were determined statistically significant.

Results

Isolation and identification of virus isolate

BYMV has a narrow host rang. Symptoms induced by the virus isolate in experimentally inoculated faba bean plants were like those expressed by field plants. BYMV induced severe and mild mosaic on all Vicia faba L. cultivars. The host range of the isolated virus was restricted to family leguminosae (Cicer arietinum, Lupinus termis, Phaseolus vulgaris, Trifolium alexandrinum and Vicia faba) and some members of Chenopodiaceae (Chenopodium album, C. amaranticolor and C. quinoa) (Figures 1&2). Stability of the isolated virus in sap indicated that, the thermal inactivation point was 60 to 65°C for BYMV, dilution end point was at 10-4 -10-5 and the longevity in vitro was 2 to 3 days. The isolated virus suggestive Bean yellow mosaic virus was mechanical transmissible readily (90–95%) with the infectious sap. Both green peach aphid (Myzus persicae Sulz.) and faba aphid (Aphis fabae) were found to be vectors of BYMV in a non-persistent manner with the average rate 80% and 40%, respectively. Seeds were harvested from mechanically inoculated faba bean (Vicia faba L.) plants and showed obvious viral infections with the isolated virus. The percentage of seed transmission was 10-13 % for BYMV in faba bean cultivar 843. BYMV was identified by ELISA. The experimental results confirmed the presence of BYMV in the infected samples. Positive reaction was obtained using BYMV-specific antiserum.

Detection of BYMV-E in infected faba bean plants was accomplished through the amplification of the viral RNA by RTPCR (Fig. 3). Isolation of RNA from plant tissues was done by SV total-RNA extraction method. The total RNA yield obtained was 137.7μg/100μl elution volume at 260nm, indicating high yield of RNA and the purity of total RNA also exhibited an A260/A280 absorbance ratio of 2.37 indicating high purity of total RNA isolated by the method described previously. Following PCR, the amplification products were analyzed by gel electrophoresis (Figure 3). The fragment was amplified from infected faba bean leaf tissues by using primer set. It appears from these data that the isolated virus is Bean yellow mosaic virus.

Effect of Bean yellow mosaic virus on faba bean growth

Chlorophyll a, b and total chlorophyll were significantly lower in infected plants (7.96, 8.37 and 15.36, respectively) compared with healthy plants (6.31, 7.13 and 13.58, respectively) at 60 days from sowing. Shoot and root length were significantly lower in infected plants compared to healthy plants. Similarly, seedling dry weight was significantly reduced due to BYMV infection in faba bean plants at 8 days after planting (Data not shown). Weight and volume of 100 seeds from infected faba bean were significantly lower compared with seeds from healthy plants. Moreover, relative density of seeds and germination percentage were also reduced in infected plants in comparison with healthy plants (Data not shown). Crude protein percentage in infected seeds was higher in infected faba bean plants (38.5) compared with the healthy seeds (33.5).

Effect of plant extracts application on BYMV disease severity

The lowest disease severity was achieved using cinnamon extracts under pot experiments. Ginger and fennel extracts (2.64, 2.81, respectively) were more effective than the extracts of clove and chamomile (3.69, 3.83, respectively). The protective activity of oil extracts was significantly effective compared with the control (8.00) (Figure 4A). On the other hand, the curative treatments of plant extracts after BYMV inoculation were also significant compared with the control (Figure 4B). Treatment with ginger extract achieved the best results (3.08) compared to the control (7.40). The lowest virus concentration was recorded in cinnamon (Cinnamomum zeylanicum) treatment (0.23) compared to control treatment (1.2). Similarly, BYMV concentration in plants treated with extracts before virus inoculation was also lower than the control (Figure 5A). Faba bean leaves sprayed with ginger extract showed the best results in this respect (0.39), compared to control treatment being 1.4 (Figure 5B).

Effect of plant extracts treatments before BYMV inoculation on the relative expression of defense genes

Our results revealed a significant (P≤0.05) increase of PR1 gene expression level in plant extract treated faba bean plants before BYMV inoculation as compared to the control (Figure 6A). Plants treated with cinnamon extracts showed the highest expression values (5.21), as compared to non-treated plants (1.01). As compared to control treatment, PR2 gene expression level was significantly increased in plant extract treated faba bean plants before BYMV inoculation (Figure 6B). In addition, plants treated with cinnamon extracts resulted in the highest upregulation of PR2 gene expression followed by fennel, chamomile, ginger, and clove (5.78, 4.56, 3.56, 2.81 and 2.28, respectively).

Effect of plant extracts treatments after BYMV inoculation on the relative expression of defense genes

Our results revealed a significant (P≤0.05) increase in PR1 gene expression level in plants treated with plant extracts as compared to the control (Figure 7A). However, no significant difference in gene expression was found between clove, chamomile, and fennel treatments (1.71, 1.69 and 1.47, respectively). When comparing all treatments, treatment with cinnamon (3.43) showed the highest expression of PR1 gene than other treatments. PR2 gene expression level was significantly increased in plants treated with plant extracts as compared to control (Figure 7B). In all groups, cinnamon and fennel treated plants (2.89 and 2.28, respectively) showed a significant higher expression than other treatments. Moreover, treatment with cinnamon resulted in higher upregulation of PR2 gene expression than fennel sprayed plants (Figure 7B).

Discussion

Recently, faba bean crop in Egypt suffered great losses due to infection with mosaic diseases. Bean yellow mosaic potyvirus (BYMV) was reported to be one of the most prevalent viruses affecting leguminous crops in Egypt. Approximately 75% of aphidvectored potyviruses are transmitted in a non-persistent (noncirculative) manner [23]. The results confirmed the presence of BYMV in the infected samples of faba bean using indirect ELISA. ELISA proved to be reliable and sensitive method for detecting and identifying BYMV in infected faba bean extracts [6,24]. Bean yellow mosaic virus (BYMV) is composed of a protein molecular weight of 30-35kd. In this study, the detection and quantification of BYMV in infected plant tissues was based on semi-quantification reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was used to detect low concentration of BYMV in total RNA extracts of infected gladiolus leaves [25].

In the present work, the decrease in growth, pigment contents and seed characters and viability probably explained based on an extensive production of excitation energy that occurred under virus stress, leading to photo-inhibitory damage in the reaction centers within chloroplasts [26]. Moreover, the lower number of chloroplasts is thought to be directly responsible for the decrease of chlorophyll content detected in BYMV-infected leaves. In compatibility with the previous results, a chloroplast is the most sensitive plant cell organelle to viral infection [27]. The application of antiviral principles derived from higher plants as biological virus control agents seems to be very promising. Antiviral proteins (AVPs) are most of these compounds, which are proteinaceous in origin.

However, deletion of the C-terminal of Pokeweed antiviral protein (PAP) mutants reduces viral infection but do not depurinate host ribosomes, indicating that PAP’s antiviral action may be separated from its ribosome inactivating proteins (RIPs) activity [28]. In the current study, faba bean plants were sprayed with plant extracts of Cinnamomum zeylanicum bark (Cinnamon), Syzygium aromaticum (Clove), Matericaria chamomilla L. (Chamomile), Foeniculum vulgare (Fennel), Zingiber officinale (Ginger) at 1 day before or after BYMV inoculation, to control BYMV in faba bean plants. Cinnamon extracts achieved the best results in reduction of disease severity, BYMV titre and activation of PR1 and PR2 gene expressions in treated plants compared with the control. Clerodendrum inerme contains basic protein which is resistant to proteases. Induced systemic resistance by antiviral agents has been introduced against many plant viruses [17,19,29,30]. A minimum concentration of 400Rg/ml of MAP (Mirabilis jalapa antiviral protein) was sufficient to inhibit TSWV [16].

In addition, Mirabilis antiviral protein (MAP) was isolated from roots and leaves of Mirabilis jalapa L. which possess repellent properties against aphids and white flies. MAP showed antiviral activity against mechanically transmitted viruses but not against aphid transmitted viruses [15]. Barakat et al. [14] reported that, Endogenous proteins have been discovered as ribosomeinactivating proteins (RIPs) such as enzymes that operate on ribosomes in a very particular manner in a variety of plant species. Six RIPs were isolated from leaves and/or seeds of several plant species, including types I and 2. Tobacco necrosis virus (TNV) on Phaseolus vulgaris plants, Tobacco mosaic virus (TMV) on Chenopodium amaranticolor plants, and Bean yellow mosaic virus (BYMV) on its systemic host (Vicia faba), all tested RIPs exhibited strong antiviral action.

Conclusion

In conclusion, the isolated virus was identified as Bean yellow mosaic virus based on host range, virus stability in crude sap, transmission, ELISA, and RT-PCR. Plant extracts was successfully used to control BYMV. The expression of pathogenesis-related genes (PR1 and PR2) was substantially higher in faba bean plants treated with plant extracts before or after BYMV inoculation which confirmed that the induced resistance was involved in disease control mechanisms.