Pilot testing of two biofungicide formulations for the control of citrus blue and green mold in two Moroccan packinghouses

Pichia guilliermondii strain Z1 and Bacillus subtilis strain ZH2 were previously selected for their high and reliable antagonistic activities against Penicillium digitatum (Sacc.) and blue mould (P. italicum Weh.) on citrus fruits. The effectiveness of pilot testing of wettable and a granular powder formulation based on each biocontrol agent was evaluated and compared to thiabendazole in two packinghouses. Each fruit was wounded on the outer surface on two opposite locations. Fruits intended to be treated with biofungicides were recovered just before applying the wax at packaging line. Fruits were soaked in a solution made with each formulation. Pathogenic inoculation was made 24 hours later by spraying two different varieties of fruits, Clementine ‘Nules’ and ‘Valencia-late’ orange, with a suspension of 105 conidia/ml of P. italicum or P. digitatum. The effect of treatment was evaluated after 7 days of fruit incubation at two temperatures 4 °C and 20 °C. The result showed that the control achieved with strain Z1 on ‘Valencia late’ orange was comparable to that with thiabendazole. On the other hand, the decay control was lower for all the treatments on the Clementine ‘Nules’. Nevertheless, ZH2 strain has no effect at 4 °C. Therefore, it was concluded that strain Z1 is a promising biocontrol agent for the control of major postharvest diseases of citrus in Moroccan packinghouse stations. Keywords: Citrus, biological control, formulation, packinghouse, Pichia guilliermondii, Bacillus subtili

Principales causes des écarts de triage des clémentines dans la région de Berkane

Citrus plants are susceptible to biotic and abiotic stresses that affect both production quality and quantity, causing high rate of grade-outs and leading to very substantial financial losses to both farmers and the packaging houses. Reduction of this rate relies on a sound knowledge of the causes of fruit rejection at packaging houses. In this context, the main causes of citrus grade-outs (clementine) have been studied in the northeast of Morocco (Berkane area). 4.606 sorting and grading forms, of the crop year 2016-2017, have been recovered from four packaging houses settled in Berkane, and have been analyzed thereafter to identify the key causes of clementine grade-outs in this area. Five categories have been identified. Physical and chemical injuries, pests, and harvest defects head the list of imperfection categories, followed by physiological disorders and fungi. In particular, the recorded grade-outs rates ranged from 5% to 65%, with an average of 27% and 30% for natural and degreened fruits, respectively. The multitude of grade-outs causes and the rapid development of resistance to pesticides commonly used in citrus orchards and packaging houses are considered as serious indicators which triggered the alarm on the necessity of searching for sustainable alternatives to protect citrus production in Berkane area.Les agrumes sont sensibles à de nombreuses contraintes biotiques et abiotiques qui affectent la production et génèrent un taux élevé des écarts de triage. La réduction de ce taux repose sur une bonne connaissance des causes de rejet des fruits au niveau des stations de conditionnement. C’est dans ce contexte que les principales causes des écarts de triage des fruits d’agrumes (cas des clémentines) ont été étudiées dans la région de Berkane. Un total de 4.606 fiches d’agréage, relevant de la campagne agricole 2016-2017, a été récupéré auprès de quatre stations de conditionnement et a été analysé profondément pour identifier les principales causes des écarts de triage des clémentines dans la région de Berkane. Les taux des écarts de triage enregistrés varient entre 5 et 65%, avec une moyenne de 27% et 30% pour les fruits de coloration naturelle et ceux déverdis, respectivement. Cinq catégories des causes des écarts de triage des clémentines ont été identifiées. Les dommages physiques et chimiques, les dommages dus aux ravageurs, et les défauts de cueillette viennent en tête des catégories des imperfections observées sur fruits, suivis par les désordres physiologiques et les infections dues aux champignons phytopathogènes. La multitude des causes des écarts de triage et le développement rapide de la résistance aux pesticides communément utilisés dans les vergers et les stations de conditionnement de Berkane sont des indicateurs déclenchant l’alarme sur la nécessité de rechercher des alternatives durables pour la protection de la production des agrumes dans la région de Berkane

Induced Systemic Resistance (ISR) in Arabidopsis thaliana by Bacillus amyloliquefaciens and Trichoderma harzianum used as seed treatments

The Trichoderma fungal species and the bacteria Bacillus species were described as inducers of plant systemic resistance in relation to their antagonistic activity. The objective of this study was to evaluate the effect of selected strains of Bacillus amyloliquefaciens (I3) and Trichoderma harzianum (A) on inducing systemic resistance in Arabidopsis thaliana as a model for plant molecular genetics. The microorganisms were identified and were confirmed for their antagonistic potential in vitro and in vivo in previous studies. In order to explore this mechanism, two mutants of A. thaliana carrying a PR1 promoter (a conventional marker of salicylic acid (SA) pathway) and LOX2 promoter (a marker triggering jasmonic acid (JA) pathway activation) were analyzed after inoculating antagonists. Transgenic reporter line analysis demonstrated that B. amyloliquefaciens I3 and T. harzianum A induce A. thaliana defense pathways by activating SA and JA at a high level compared to lines treated with chemical elicitors of references (acibenzolar-S-methyl (Bion 50 WG (water-dispersible granule)), SA, and methyl jasmonate). The efficacy of B. amyloliquefaciens I3 and T. harzianum A in inducing the defense mechanism in A. thaliana was demonstrated in this study

Evaluation of antagonistic potential of some isolates of bacteria and Trichoderma spp. in the control of Zymoseptoria tritici causal agent of wheat leaf spot

The aim of this study is to develop a bio-control method against Zymoseptoria tritici blotch (STB) which is a leaf disease caused by the fungus Zymoseptoria tritici in wheat. This disease in one the main diseases of wheat in Morocco. 120 isolates of bacteria and 70 isolates of Trichoderma spp. of various ecological and geographical origins, were tested in vitro for their antagonistic potential towards two strains of Zymoseptoria tritici, namely G1-1 which was isolated from soft wheat (Triticum aestivum L.) and A5-1 which was isolated from durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.). Compared to the controls, all the antagonists selected after the in vitro tests (9 bacterial strains and 18 Trichoderma spp. isolates) showed a very high antagonism potential, generating zones of inhibition of the radial growth of Zymoseptoria tritici, which ranged from 34 to 84 % for the bacterial strains and from 40 to 70 % for the Trichoderma spp., after 96 hours of confrontation. To confirm the performance of the selected isolates in vitro, an in vivo assay on inoculated potted plants by the pathogen was conducted using two sensitive wheat varieties to septoria disease; ‘Aguilal’ (soft wheat variety) and ‘Karim’ (durum wheat variety). Spraying of selected antagonists suspensions onto leaf surfaces of wheat plants proved to be effective and significantly reduced the severity of the septoria disease up to 91 % in the preventive treatments. For curative treatments, the intensity of disease was more important. The symptoms were reduced from 7 to 72 % in the case of bacterial treatments and from 19 to 81 % for Trichoderma spp. strains

Evaluation of antagonistic potential of some isolates of bacteria and Trichoderma spp. in the control of Zymoseptoria tritici causal agent of wheat leaf spot

Le but de cette étude est de développer une méthode de lutte biologique contre la septoriose (STB) qui est l’une des principales maladies foliaires du blé au Maroc, causée par le champignon Zymoseptoria tritici. 120 isolats de bactéries et 70 isolats de Trichoderma spp. de différentes origines écologiques et géographiques, ont été testés in vitro pour leur potentiel antagoniste vis-à-vis de deux souches de Zymoseptoria tritici à savoir G1-1, dont l’une a été isolée du blé tendre (Triticum aestivum L.) et l’autre A5-1 du blé dur (Triticum turgidum L. subsp., durum (Desf.) Husn.). Comparés aux témoins, tous les antagonistes sélectionnés suite aux tests in vitro (9 souches bactériennes et 18 isolats de Trichoderma spp.) ont montré un potentiel d’antagonisme très élevé, avec des zones d’inhibition de la croissance radiale de Zymoseptoria tritici allant de 34 à 84 % pour les souches bactériennes et de 40 à 70 % pour les Trichoderma spp., après 96 heures de confrontation. Pour confirmer la performance des isolats sélectionnés suite aux tests in vitro, des tests in vivo sur des plants de blé en pots ont été conduits moyennant deux variétés de blé sensibles à la septoriose, à savoir ‘Aguilal’ (variété de blé tendre) et ‘Karim’ (variété de blé dur). Les pulvérisations foliaires de suspensions d’antagonistes sélectionnés se sont révélées efficaces et ont réduit significativement la sévérité de la maladie jusqu’à 91 % dans les traitements préventifs. Pour les traitements curatifs, la sévérité de la septoriose était plus importante. En effet, la réduction des symptômes varie de 7 à 72 % dans le cas des traitements à base de souches de bactéries et de 19 à 81 % pour ceux à base de Trichoderma spp. Mots-clés: Zymoseptoria tritici, Trichoderma spp., Bactéries, lutte biologique, blé.The aim of this study is to develop a bio-control method against Zymoseptoria tritici blotch (STB) which is a leaf disease caused by the fungus Zymoseptoria tritici in wheat. This disease in one the main diseases of wheat in Morocco. 120 isolates of bacteria and 70 isolates of Trichoderma spp. of various ecological and geographical origins, were tested in vitro for their antagonistic potential towards two strains of Zymoseptoria tritici, namely G1-1 which was isolated from soft wheat (Triticum aestivum L.) and A5-1 which was isolated from durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.). Compared to the controls, all the antagonists selected after the in vitro tests (9 bacterial strains and 18 Trichoderma spp. isolates) showed a very high antagonism potential, generating zones of inhibition of the radial growth of Zymoseptoria tritici, which ranged from 34 to 84 % for the bacterial strains and from 40 to 70 % for the Trichoderma spp., after 96 hours of confrontation. To confirm the performance of the selected isolates in vitro, an in vivo assay on inoculated potted plants by the pathogen was conducted using two sensitive wheat varieties to septoria disease; ‘Aguilal’ (soft wheat variety) and ‘Karim’ (durum wheat variety). Spraying of selected antagonists suspensions onto leaf surfaces of wheat plants proved to be effective and significantly reduced the severity of the septoria disease up to 91 % in the preventive treatments. For curative treatments, the intensity of disease was more important. The symptoms were reduced from 7 to 72 % in the case of bacterial treatments and from 19 to 81 % for Trichoderma spp. strains. Keywords: Zymoseptoria tritici, Trichoderma spp., Bacteria, biological control, wheat

Effects of Temperature and Relative Humidity on the In Vitro and In Vivo Radial Growth of Penicillium italicum and on the Biocontrol Activity of Pichia guilliermondii, Strain Z1

peer reviewedThe objective of this study was to assess the effect of temperature (5-25°C) on the ‘in vitro’ and ‘in vivo’ growth rates of Penicillium italicum and to determine the combined effect of temperature and relative humidity (45 to 100%) on lesion size of this pathogenic fungus on Valencia late oranges, either alone or in combination with the antagonistic yeast strain Z1 of Pichia guilliermondii Wickerham. Statistical analysis showed a significant effect of temperature on the ‘in vitro’ and ‘in vivo’ radial growth of P. italicum with the maximum growth observed at temperature of 25°C. In both cases, no growth was observed at a temperature of 35°C. These factors had a significant effect on P. italicum lesion size when it was applied alone on Valencia late oranges and insignificant when yeast strain Z1 was applied 24 h before P. italicum inoculation. Our results confirm previous ‘in vitro’ findings that aw has a greater influence than temperature on P. italicum growth and highlight that the strain Z1 showed high antagonistic potential against this pathogen over a range of temperature-relative humidity regimes favouring P. italicum development

Principales causes des écarts de triage des clémentines dans la région de Berkane

Citrus plants are susceptible to biotic and abiotic stresses that affect both production quality and quantity, causing high rate of grade-outs and leading to very substantial financial losses to both farmers and the packaging houses. Reduction of this rate relies on a sound knowledge of the causes of fruit rejection at packaging houses. In this context, the main causes of citrus grade-outs (clementine) have been studied in the northeast of Morocco (Berkane area). 4.606 sorting and grading forms, of the crop year 2016-2017, have been recovered from four packaging houses settled in Berkane, and have been analyzed thereafter to identify the key causes of clementine grade-outs in this area. Five categories have been identified. Physical and chemical injuries, pests, and harvest defects head the list of imperfection categories, followed by physiological disorders and fungi. In particular, the recorded grade-outs rates ranged from 5% to 65%, with an average of 27% and 30% for natural and degreened fruits, respectively. The multitude of grade-outs causes and the rapid development of resistance to pesticides commonly used in citrus orchards and packaging houses are considered as serious indicators which triggered the alarm on the necessity of searching for sustainable alternatives to protect citrus production in Berkane area.Les agrumes sont sensibles à de nombreuses contraintes biotiques et abiotiques qui affectent la production et génèrent un taux élevé des écarts de triage. La réduction de ce taux repose sur une bonne connaissance des causes de rejet des fruits au niveau des stations de conditionnement. C’est dans ce contexte que les principales causes des écarts de triage des fruits d’agrumes (cas des clémentines) ont été étudiées dans la région de Berkane. Un total de 4.606 fiches d’agréage, relevant de la campagne agricole 2016-2017, a été récupéré auprès de quatre stations de conditionnement et a été analysé profondément pour identifier les principales causes des écarts de triage des clémentines dans la région de Berkane. Les taux des écarts de triage enregistrés varient entre 5 et 65%, avec une moyenne de 27% et 30% pour les fruits de coloration naturelle et ceux déverdis, respectivement. Cinq catégories des causes des écarts de triage des clémentines ont été identifiées. Les dommages physiques et chimiques, les dommages dus aux ravageurs, et les défauts de cueillette viennent en tête des catégories des imperfections observées sur fruits, suivis par les désordres physiologiques et les infections dues aux champignons phytopathogènes. La multitude des causes des écarts de triage et le développement rapide de la résistance aux pesticides communément utilisés dans les vergers et les stations de conditionnement de Berkane sont des indicateurs déclenchant l’alarme sur la nécessité de rechercher des alternatives durables pour la protection de la production des agrumes dans la région de Berkane

Effets du porte-greffe sur le rendement et la qualité des fruits de la mandarine «Nadorcott»

The effects of citrus rootstocks on the performance and fruits characteristics of commercial varieties are known and well documented. These effects, as well as the resistance to biotic and abiotic stresses, constitute the main criteria that direct the rootstock choice for a new citrus orchard. The Moroccan variety ‘Nadorcott’ mandarin also called ‘W. Murcott’ and ‘Afourer’ is relatively new. ‘Nadorcott’ is a popular late mandarin cultivar, owing to its good eating quality and excellent bearing capacity. Thus, there are few undertaken research and available results related to its behavior on the widely used rootstocks in the main citrus growing areas of Morocco Kingdom. This paper presents the results of a field trial carried out in the North West Morocco, a coastal area, to evaluate the effects of five rootstocks on ‘Nadorcott’ yield and fruit quality: juice content, total soluble solids (TSS), acid content (A), maturity index expressed as TSS/A ratio and Citrus Color Index (CCI). The results show that the highest yields were observed on citrange C-35 and Citrus macrophylla (MAC) over the three seasons. Additionally, percentage of juice content over two successive years of fruits produced on C-35, Troyer citrange (CT), Carrizo citrange (CC) and Swingle citrumelo (SW) was about 53 % but did not exceed 50 % for Citrus macrophylla. The highest TSS was obtained on CC rootstock. However, the TSS was less than 10 in 2013 and around 11 °Brix in 2014 for Citrus macrophylla and above 12 °Brix for the other rootstocks. Regarding the maturity index, apart from the low value (11.3) scored by MAC, in 2013 due to a low TSS, there was no significant difference between the maturity indices of the fruits of the mandarin 'Nadorcott' produced on the different rootstocks tested. Furthermore, the five rootstocks had no effect on the fruit color parameters of 'Nadorcott' mandarin.Les effets des porte-greffes d'agrumes sur les performances et les caractéristiques des fruits des variétés commerciales sont connus et bien documentés. Ces effets, ainsi que la résistance aux stress biotiques et abiotiques, constituent les principaux critères qui orientent le choix d’un porte-greffe pour la mise en place d’un nouveau verger d'agrumes. La variété marocaine de mandarine «Nadorcott» également appelée «W. Murcott » ou « Afourer » un mandarinier tardif populaire, en raison de sa bonne qualité gustative et de son excellente capacité productive. Peu de recherche ont été entreprises et peu de résultats sont disponibles sur son comportement en association avec les porte-greffes largement utilisés dans les principales zones de production d'agrumes au Maroc. Cet article, présente les résultats d'un essai porte-greffe installé dans le nord-ouest du Maroc, une zone côtière, dans le but d’évaluer les effets de cinq porte-greffes sur le rendement de la 'Nadorcott' et sur la qualité des fruits notamment la teneur en jus, les sucres solubles totaux (TSS), l’acidité (A), l’indice de maturité exprimé par le rapport (TSS/A) et sur la coloration de l’écorce exprimée par Citrus Color Index (CCI). Les résultats obtenus montrent que les rendements les plus élevés, au cours des trois saisons, ont été réalisés par le citrange C-35 et le Citrus macrophylla (MAC). En outre, le pourcentage de teneur en jus, sur deux années consécutives, des fruits produits avec le C-35, le citrange ‘Troyer’ (CT), le citrange ‘Carrizo’ (CC) et le citrumelo ‘Swingle’ (SW) était d'environ 53% mais il ne dépassait pas 50 % pour le Citrus macrophylla. Le TSS le plus élevé a été obtenu avec le porte-greffe CC. Cependant, le TSS était inférieur à 10 en 2013 et autour de 11 °Brix en 2014 pour le Citrus macrophylla et il était supérieur à 12 °Brix pour les autres porte-greffes. Concernant l'indice de maturité, hormis la valeur de (11,3) notée par MAC, en 2013 en raison d'un TSS faible, il n'y avait pas de différence significative entre les indices de maturité des fruits du mandarin 'Nadorcott' produits sur les différents porte-greffes testés. De plus, les cinq porte-greffes n'ont eu aucun effet sur les paramètres de couleur des fruits du mandarinier 'Nadorcott'

Control of citrus blue mold by the antagonist yeast Pichia guilliermondii Z1: compatibility with commercial fruit waxes and putative mechanisms of action.

Pichia guilliermondii strain Z1, which was previously proven to be effective against blue mold of citrus fruit, has been further tested in controlled conditions to determine whether the yeast, as an alternative for synthetic fungicides, would be compatible with other postharvest practices used commercially. In particular, commercial fruit waxes can reduce their survival and effectiveness. The commercial fruit waxes tested, in combination at 20% with strain Z1, included microcrystalline, ester gum, candelilla, beeswax, montan, paraffin, rice bran, rosin maleic, carnauba, shellac, and one mixture shellac plus carnauba. Beeswax, paraffin, rosin maleic, carnauba, and shellac increased significantly strain Z1 survival in Petri dish assays. Candelilla, beeswax, rice bran, rosin maleic, carnauba, shellac, and shellacecarnauba mixture did not significantly reduce the strain Z1 yield on orange fruit surfaces compared to other waxes. With the exception of rosin maleic wax, none of the commercial fruit waxes or mixture increased significantly the ability of the formulated product of strain Z1 to control the postharvest pathogen Penicillium italicum on wounded orange fruit. When the formulated product of strain Z1 was used in combination with beeswax, strain Z1 retained the same efficacy. In contrast, microcrystalline, ester gum, candelilla, montan, paraffin, rice bran, carnauba, shellac, and shellac-carnauba mixture significantly reduced the effectiveness of the formulated product of strain Z1. No antibiosis was detected for strain Z1 against P. italicum. Strain Z1 inhibited the spore germination for the low juice concentration (up to 5%) when compared to the control. However, the addition of fresh juice after antagonist removal allowed the restoration of the germination of P. italicum spores; suggesting the possibility of competition for nutrients in the biocontrol activity of strain Z1. The study demonstrates the potential commercial application of strain Z1 with beeswax and rosin maleic wax for postharvest control of citrus blue mold. As biocontrol relies on competition for nutrients, an enriched formulation with nutrients is needed for reliable antifungal activity of this yeast strai