Effects of plant growth promoting rhizobacteria (PGPR) on Citrus macrophylla rootstock

Citrus is one of the largest fruit crops grown in Morocco. Citrus crops gain in importance due to the jobs generated during the production process of fresh or processed fruit. Intensive agriculture is characterized by the excessive use of inorganic fertilizers and pesticides. This production system has generated serious environmental contamination problems, thus, it is necessary to implement sustainable production strategies to reduce the use of synthetic chemicals and contribute to soil and water conservation. In this context, Seventy two Rhizobacterial isolates of fluorescent Pseudomonas were isolated from rhizosphere soil of Citrus in the Sapiama nursery. These isolates were tested on germination and growth of Citrus macrophylla rootstock. The results obtained showed that the isolate C11 significantly stimulated germination 16 days after seed inoculation. The C26, C6 and C24 isolates showed PGPR effects improving significantly the growth parameters of C. macrophylla rootstock. They significantly promoted plant height, collar diameter and root length. This study concluded that the Pseudomonas isolates could be potential alternative biofertilizers to chemical products and could be considered as a promising main component for sustainable agriculture development strategy in Citrus farming. Keywords: Citrus macrophylla, Pseudomonas, PGP

Rhizospheric solutions: Pseudomonas isolates counter Botrytis cinerea on tomato

La moisissure grise causée par Botrytis cinerea provoque des dégâts sur plus de 200 espèces de cultures dans le monde. B. cinerea sporule pour former une pourriture grise sur les feuilles, les tiges et les fruits. Pour lutter contre B. cinerea, des fongicides synthétiques sont utilisés. Ces derniers mettent en danger la santé humaine et environnementale en plus de la résistance qu'ils peuvent occasionner chez les souches de B. cinerea. Les alternatives écologiques sont des solutions appropriées pour contrôler la moisissure grise tout en maintenant l’équilibre environnemental. L’objectif de cette étude est d'évaluer l’effet des isolats de Pseudomonas issus de la rhizosphère de la tomate sur B. cinerea. Les résultats ont montré que les 76 isolats testés inhibent le développement de B. cinerea in vitro. Cinq isolats de Pseudomonas (Q6B, Q13B, Q7B, Q14B et Q1B) ont provoqué des niveaux d'inhibition significatifs allant de 65 à 73%. Par ailleurs, ces isolats ont également inhibé B. cinerea sur les feuilles et le fruit de la tomate. Pour tenter d'élucider les mécanismes d'action, les cinq isolats ont montré une production des métabolites antifongiques tels que les sidérophores, le cyanure d'hydrogène et d’autres enzymes. Les résultats de cette étude ont montré que les isolats de Pseudomonas Q6B, Q13B, Q7B, Q14B et Q1B ont une forte efficacité dans la lutte biologique contre B. cinerea et peuvent être utilisés pour une lutte écologique durable.Gray mold caused by Botrytis cinerea causes serious losses in more than 200 crop species worldwide. The necrotrophic fungus sporulates to effect a grey covering on leaves, stems and flowers. B. cinerea is controlled by chemical synthetic fungicides, endangering human and environmental health. Synthetic fungicides stimulate emergence of pathogen resistance. Organic alternatives which may be present or introduced into the edaphic environment are suitable solutions to control outbreaks. This study was done in order to elucidate the mode of action involved in the control of B. cinerea using fluorescent Pseudomonas isolates from tomato roots. The results show that all 76 isolates inhibit fungal growth during in vitro bioassay using dual culture technique. Five isolates of Pseudomonas (Q6B, Q13B, Q7B, Q14B and Q1B) cause significant inhibition levels ranging from 65 to 73%. These isolates inhibit fungal growth in both fruits and leaves. Each isolate tested produced antifungal metabolites (siderophores, hydrogen cyanide and enzymes). Results of this study show that all tested Pseudomonas isolates have a strong efficacy in biological control against B. cinerea and can be used for environmentally sustainable control

First Report of Little Cherry Virus 1 Infecting Apricot (Prunus armeniaca) in Africa

peer reviewedLittle cherry disease (LChD) is an important viral disease of many stone fruit species (Prunus spp.), sweet cherry (Prunus avium L.) being the most common host. It is associated with two different virus species belonging to the family Closteroviridae, namely, Little cherry virus 1 (LChV-1, Velarivirus) and Little cherry virus 2 (LChV-2, Ampelovirus). The impact of LChD on sweet cherry production consists in the decrease of yield and fruit quality, which is mainly associated with LChV-2, whereas most of LChV-1 reported infections remain associated with an unclear etiology. Other stone fruit species, such as peach and plum, hosting LChV-1 have been reported (Matic et al. 2007; Šafářová et al. 2017). LChV-1 is mainly transmitted through propagation of infected plant material, and no vector transmission is known (Jelkmann and Eastwell 2011). In 2018, during the early vegetative season, a limited survey was carried out for virus detection in apricot and sweet cherry orchards in the main southern Moroccan stone fruit-producing regions of Agadir, Agdez, and Dayat Aoua. Two sweet cherry trees (P. avium ‘Coeur de Pigeon’ and ‘Bigarreau’) and three apricot trees (Prunus armeniaca L.), all asymptomatic, were sampled (five branches with leaves) from three different orchards. RNA was extracted (both leaves and cambial scrapings) using the Spectrum Plant Total RNA kit (Sigma-Aldrich, Belgium), prior to cDNA synthesis using the iScript Reverse Transcription Kit (Bio-Rad, Belgium). LChV-1 detection was done by reverse transcription PCR (RT-PCR) using the specific primers LCUW7090 (5′-GGTTGTCCTCGGTTGATTAC-3′)/LCUWc7389 (5′-GGCTTGGTTCCATACATCTC-3′) (Bajet et al. 2008), amplifying a 300-bp fragment spanning the ORF1b encoding the RdRp gene, and 1LC_12776F (5′-TCAAGAAAAGTTCTGGTGTGC-3′)/1LC_13223R (5′-CGAGCTAGACGTATCAGTATC-3′) (Glasa et al. 2015), targeting a 456-bp fragment of the CP gene. LChV-2 specific primers were used according to Eastwell and Bernardy (2001). RT-PCR results revealed the presence of LChV-1 in two apricot samples from Agdez. No LChV-1 was detected in the sweet cherry samples. The presence of LChV-1 was confirmed by means of the LChV-1 specific reverse transcription loop-mediated isothermal amplification approach as described by Tahzima et al. (2019). No LChV-2 was detected in any of the samples. The RdRp and CP specific amplification products were bidirectionally sequenced (Genewiz, Leipzig, Germany) and assembled. RdRp and CP partial nucleotide sequences of the Moroccan LChV-1 isolates MOT2 and MOA1 were deposited in GenBank (accession nos. MK905349, MK905350; and MK905351, MK905352, respectively). Based on BLAST analysis of RdRp and CP, the Moroccan LChV-1 sequences shared 99% nucleotide identity (99.55% amino acids) with the No2ISTO isolate (HG792418) from Greece and 97.96% (98.64% amino acids) with the Spanish Ponferrada isolate (KX192367), respectively. Although the presence of LChV-1 has previously been reported in many countries in different continents, to our knowledge, this represents the first detection of LChV-1 in Africa

First Report of Apricot vein clearing-associated virus (AVCaV) infecting Prunus domestica revealed by High-Throughput Sequencing in Africa

Plum (Prunus domestica L., Rosaceae) trees, like many stone fruit trees, are known to be infected by numerous plant viruses, predominantly as consequence of their clonal mode of propagation and perennial cultivation (Jelkmann and Eastwell, 2011). Apricot vein clearing-associated virus (AVCaV) is a member of the genus Prunevirus in the family Betaflexiviridae. AVCaV was first reported in Italy infecting apricot (P. armeniaca L.) associated with foliar vein clearing symptoms (Elbeaino et al. 2014). It has also been detected in various Prunus species, like plum, Japanese plum (P. salicina L.), sour cherry (P. cerasus L.), and Japanese apricot (P. mume L.), apricot and peach (P. persica L.) sourced from Asian and European countries (Marais et al. 2015), as well as in the ornamental Myrobolan plum (P. cerasifera L.) in Australia (Kinoti et al. 2017). In 2018, during the vegetative season, a survey was carried out in two different apricot and plum orchards in the southern region of Agdez (Agadir, Morocco) where stone fruit trees are grown. Five branches with leaves were sampled from three apricot and three plum trees of unknown cultivars, all asymptomatic. Total RNA was extracted from 100 mg plant tissue (leaves and cambial scrapping) using RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and separate samples (one per species) were used for library preparation (NEBNext Ultra RNA library kit; New England BioLabs, MA, USA), and sequencing (Illumina NextSeq v2, totRNA sequencing) at Admera Health (New Jersey, USA). All generated reads (6,756,881) from the plum sample were quality filtered and submitted to the VirusDetect pipeline (Zheng et al., 2017). The plum cDNA library, a total of 20 viral contigs (68-1928 bp) mapped to several AVCaV accessions in GenBank. A reference mapping (CLC Genomics Workbench 12, Qiagen, Denmark) was conducted against all four available AVCaV full genomes (KM507062-63, KY132099 and HG008921), revealing 100% coverage of the full sequence (8358 nt) with 97-98 % nucleotide (nt) identities (BLASTn). Analysis of the derived sequences allowed to identify the location of the four predicted ORFs i.e. (ORF1: 6066 nt/2,021 aa), (ORF2: 1383 nt/460 aa), (ORF3: 666 nt/221 aa) and (ORF4: 420 nt/139 aa), previously described for the AVCaV genome (Elbeaino et al. 2014). The amino acid sequences of the encoded proteins of AVCaV isolate from Morocco also shared 97-98% identities with the corresponding sequences of complete genome AVCaV isolates in GenBank. To confirm the detection of AVCaV in the three plum samples, specific RT-PCR primers (VC37657s: 5’-CCATAGCCACCCTTTTTCAA-3’ / VC28239a: 5’-GTCGTCAAGGGTCCAGTGAT-3’) (Elbeaino et al. 2014) were used and the expected 330 bp fragment from the replicase gene was amplified in all three samples and subsequently sequenced (MT980794-96). Sanger sequences were 100% identical to corresponding HTS derived sequence. This is the first report of AVCaV infecting plum in Africa. The incidence of AVCaV in Moroccan Prunus species is unknown. Plum trees from the surveyed orchards were also confirmed to be co-infected with little cherry virus 1 (LChV-1) using HTS. Further investigation is required to determine the impact of AVCaV on these asymptomatic plum trees and other stone fruits species

Solutions rhizosphériques : Isolats de Pseudomonas contre Botrytis cinerea de la tomate

Gray mold caused by Botrytis cinerea causes serious losses in more than 200 crop species worldwide. The necrotrophic fungus sporulates to effect a grey covering on leaves, stems and flowers. B. cinerea is controlled by chemical synthetic fungicides, endangering human and environmental health. Synthetic fungicides stimulate emergence of pathogen resistance. Organic alternatives which may be present or introduced into the edaphic environment are suitable solutions to control outbreaks. This study was done in order to elucidate the mode of action involved in the control of B. cinerea using fluorescent Pseudomonas isolates from tomato roots. The results show that all 76 isolates inhibit fungal growth during in vitro bioassay using dual culture technique. Five isolates of Pseudomonas (Q6B, Q13B, Q7B, Q14B and Q1B) cause significant inhibition levels ranging from 65 to 73%. These isolates inhibit fungal growth in both fruits and leaves. Each isolate tested produced antifungal metabolites (siderophores, hydrogen cyanide and enzymes). Results of this study show that all tested Pseudomonas isolates have a strong efficacy in biological control against B. cinerea and can be used for environmentally sustainable control.La moisissure grise causée par Botrytis cinerea provoque des dégâts sur plus de 200 espèces de cultures dans le monde. B. cinerea sporule pour former une pourriture grise sur les feuilles, les tiges et les fruits. Pour lutter contre B. cinerea, des fongicides synthétiques sont utilisés. Ces derniers mettent en danger la santé humaine et environnementale en plus de la résistance qu'ils peuvent occasionner chez les souches de B. cinerea. Les alternatives écologiques sont des solutions appropriées pour contrôler la moisissure grise tout en maintenant l’équilibre environnemental. L’objectif de cette étude est d'évaluer l’effet des isolats de Pseudomonas issus de la rhizosphère de la tomate sur B. cinerea. Les résultats ont montré que les 76 isolats testés inhibent le développement de B. cinerea in vitro. Cinq isolats de Pseudomonas (Q6B, Q13B, Q7B, Q14B et Q1B) ont provoqué des niveaux d'inhibition significatifs allant de 65 à 73%. Par ailleurs, ces isolats ont également inhibé B. cinerea sur les feuilles et le fruit de la tomate. Pour tenter d'élucider les mécanismes d'action, les cinq isolats ont montré une production des métabolites antifongiques tels que les sidérophores, le cyanure d'hydrogène et d’autres enzymes. Les résultats de cette étude ont montré que les isolats de Pseudomonas Q6B, Q13B, Q7B, Q14B et Q1B ont une forte efficacité dans la lutte biologique contre B. cinerea et peuvent être utilisés pour une lutte écologique durable