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Recent Doctorate Theses in Plant Protection (2017/18)


Anissa, Lahdhiri. 2017. Weed Flora Diagnosis of crop systems. Doctorate Thesis in Sustainable Agriculture, ISA Chott-Meriem, University of Sousse, Sousse, Tunisia, 189 pp. (Public Defense: 30 September 2017)

The agronomic diagnosis of the cropping systems (CS) is a crucial step, allowing their evaluation and innovation. This study is a contribution to reflection to enhance sustainability of CS in the irrigated public area of Chott-Meriem (IPA). The objective is to analyze the weed flora as an assessment indicator of CS in the Agro-ecosystem in this IPA. Firstly, we studied weed densities detected throughout the long-term experiment on cereal CS at the INRA of Dijon-Epoisses farm. Principal component analyzes (PCA) concluded that the weed flora composition is correlated with the cultural practices characterizing each CS. This result confirms our hypothesis that weeds can be a relevant indicator of CS durability.

Involving weed flora in the diagnosis requires a specific protocol, especially concerning the choice of optimal sampling unit size. Among the five studied sizes (0.05 m2, 0.1 m2, 0.2 m2, 0.4 m2 and 0.8 m2), we concluded that the quadrat of 0.2 m2 provides reliable results at a lower cost. Monitoring the evolution of weeds in three forage crops (berseem (Trifolium alexandrinum), ryegrass (Lolium multiflorum) and oats (Avena sativa)) showed that berseem was the least weeded with 57, 37 and 24 weeds/m2 in the first three surveys due to its post-emergent growth rate and its ability to fix atmospheric nitrogen. Weeds accounted for 43 and 48% of the total vegetation in oats respectively at the second and third surveys. This weed competition had a negative impact on oat biomass, which is shown by Pearson's negative correlation between weed density and oat biomass (r = -0.664, P = 0.003). The high weeding of the three crops is correlated with their weakening caused by their low establishment and neglect of their cultural preferences.

In a third step, we lead off the agronomic diagnosis of zucchini (Cucurbita pepo) crop management in the IPA of Chott-Meriem. Through regular monitoring of farmers, we recorded seven descriptors that are: previous crop, planting, irrigation, fertilization, weed control, crop control and yield. The unsuitable plantation date, the random irrigation and crop control, the low fertilizer inputs (11-68% N, 2-126% of P2O5 and >11% K2O) and the inadequate rotation explain the low yields (<33 t/ha). As for the agroecological diagnosis, it was based on periodic and stratified weed surveys whose analysis shows the dominance of the Therophytes species characteristic of disturbed areas. The Shannon Diversity Index has been low (<2) reflecting undiversified communities. Weed densities were high with maximums of 157, 58 and 77 plants/m2 respectively on the edge, the row and the inter-row reflecting the failure of current crop management to control these communities. The results of the agronomic and agroecological diagnoses converge to show the crop management fragility causing the CS failure in the IPA. The rational planning of crop management, at the plot scale, is a basic step to innovate these CS and promote their sustainability.

Ultimately, this study, with methods and obtained results, presents an interesting methodological reflection that deserves to be extrapolated at the succession scale and at the level of other IPA for innovation.

Cherif, Asma. 2018. The tomato leafminer Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae): Biological, ecological parameters and alternative control methods. Doctorate thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 182 pp. (Public Defense: 11 January 2018)

Tuta absoluta (Lepidoptera: Gelechiidae) is an important pest of tomato crops causing significant yield losses up to 80% in newly invaded area. Obtained results of this study indicated a high genetic conformity of this pest and its susceptibility to develop in tested artificial diets. Our data showed that this pest was able to develop under different levels of temperature (21, 25 and 28°C) and relative humidity (32, 52 and 72%) as well as various host plants (tomato, potato and eggplants). This pest was able to achieve up to 4-5 flight peaks and 3-4 generations of eggs and larvae under greenhouse and field conditions. A positive correlation between some specific parameters (captured males and laid eggs, captured males and mines with larvae, captured males and total mines and mines with larvae and mines without larvae) was emphasized. The combined use of mass trapping and insecticides gave encouraging results given that problems of resistances were not found. Dose of twenty Trichogramma cocoeciae (Hymenoptera: Trichogrammatidae) tested in protected and open filed crops was the most effective in reducing the pest population. This study shows the toxicity of some insecticides (indoxacarb, spinosad…), widely used in Tunisia tomato crops, on all T. cocoeciae development stages and highlights the safety of others (azadirachtin, Bacillus thuringiensis and virus HaNPV). This study allows a better understanding of T. absoluta issue in terms of biology, population dynamics and genetic characterization. It proposes efficient control strategies when using effective insecticides with less side effects on parasitoid. Also, this research promotes a better biological control using Trichogramma mass releases. However, additional studies, are still required to test new control strategies and propose new ones in order to avoid problems of resistance as reported for example in many countries such as in Brazil.

Cherif, Amira. 2018. Study of the bio-ecology of cereal midges and assessment of some control methods. Doctorate Thesis in Agronomic Sciences (Phytiatry), INAT, University of Carthage, Tunis, Tunisia, 140 pp. (Public Defense: 28 February 2018)

Cereal midges are considered among the most destructive pests of wheat, barley and oat in most cereal areas of the world. These pests cause significant economic losses to cereal yields. During the present work, a survey was carried out, in north Tunisia, in order to identify and study the distribution of the different gall midge species in this area. The study of the bio-ecology of these insect pests, the impact of their damages and the evaluation of some pest control methods against these midges were also the objectives of this work.

Through a molecular characterization, using two sequences of the mitochondrial DNA the cytochrome oxidase c and the 16S rDNA genes, three species of gall midges were recognized as infesting cereals in north Tunisia: wheat gall midge or Hessian fly Mayetiola destructor, barley stem gall midge Mayetiola hordei and oat gall midge Mayetiol aavenae.

These species of midges are widely distributed in all surveyed cereal areas of north Tunisia: Bizerte, Beja, Zaghouan, Siliana and Kef, but with frequencies, infestation percentages and incidence rates higher in semi-arid regions than in sub-humid ones. During the 3 years study, highest infestations were observed in the region of Zaghouan. On the other hand, M. hordei has proved to be the most predominant gall midge in north Tunisia.

Populations’ dynamic of the 3 gall midge species showed that adults’ emergence, population densities and the duration of each developmental stage differed depending on species, years, and regions. These differences are highly dependent on climatic factors, mainly temperature and rainfall. Indeed, heavy rainfalls (more than 70 mm) could reduce gall midges’ density. Likewise, low and high temperatures may decrease the emergence density of gall midge adults as observed with M. destructor. However, the number of generations and the life cycle of these insects remain basically the same. Indeed, for the three gall midge species M. destructor, M. hordei and M. avenae, 3 generations were detected annually on wheat, barley and oat crops. The first and second generations are complete and the last one is incomplete since no emerged adults were observed. Indeed, once temperatures become high (from 23°C), cereal gall midges enter in diapause and spend the summer as pupae

Assessment of gall midges impact on cereal’s yield parameters has shown that for all cereal species, plant height was the most affected parameter followed by tillers number and shoot dry weight. Study of the co-occurrence of M. hordei and M. destructor on barley crops showed that M. hordei is present during all the year on barley, whereas M. destructor is only observed from March month. In a final part, the assessment of some control methods to minimize populations of these insect pests was performed.

Trials to identify sources of resistance showed that varieties containing H22, H25 and H26 genes are resistant to the Tunisian biotype of Hessian fly. On the other hand, the barley variety Kounouz was the least attacked by M. hordei compared to the other tested varieties. For crop control, our study showed that seasonal seeding of wheat and barley is recommended to minimize midge populations. Indeed, the later is the planting date, the greater are infestation percentages, incidence rates and damages of gall midges on cereal plants. On the other hand, a biennial rotation with a no-till sowing of wheat was a good combination to reduce infestation percentages of durum wheat by midge. The results obtained in the present study could be used to develop an integrated pest management program against cereal midges in Tunisia.

Nefzi, Ahlem. 2018. Valorization of two wild Solanaceae (Solanum linnaeanum and Lycium arabicum) as potential sources of bioactive molecules and biocontrol agents against Fusarium oxysporum f. sp. radicis-lycopersici infecting tomato. Doctorate Thesis in Biological Sciences, Faculty of Sciences of Bizerte, University of Carthage, Tunisia, 171 pp. (Public Defense: 17 April 2018)

As a part of the research for alternatives to the industrial products, the present work aims the valuation of endophytic fungi and plant extracts from two Solanaceae species (Solanum linnaeanum and Lycium arabicum) on tomato cv. Rio Grande plants to control Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and to enhance tomato growth. The approach consists in the collection of fungal isolates from both species and the study of their endophytic capacity in tomato. Fungal isolates (used as conidial suspensions or cell-free culture filtrates), were shown able to colonize tomato tissues, as well as aqueous (15 and 30% w/v) and organic (chloroform, ethyl acetate and butanol) extracts used at 1, 2, 3 and 4% v/v were screened for their growth-promoting effect in tomato and their in vivo and in vitro antifungal potential against FORL. Results showed that, among 115 fungal isolates, recovered from the sterilized surfaces of leaves, stems, flowers and fruits from both species, 15 isolates presented similar morphological traits as the wild types and were considered as endophytes. Tested under greenhouse conditions, the isolates I74, I92, I15 and I18 were found to be the most active in improving root elongation by 83.8-91.8%, plant height by 80.5-94%, roots fresh weight by 91.3%, and aerial part fresh weight by 84-91.6% in treated tomato plants as compared to the untreated controls. These endophytes were the most bioactive in the suppression of disease severity by reducing the leaf and root alteration index by 80-90%, the vascular browning extent by 84-97.2 and FORL colony diameter by 77.2-82.1% compared to FORL-inoculated and hymexazol-treated controls. The molecular identification of the most bioactive isolates allowed their affiliation to four fungal species namely Penicillium crustosum (MF188258), Fusarium proliferatum (MF188256), Alternaria alternata (MF693801), and F. proliferatum (MF693802). Leaf aqueous extracts from both species, applied at 30% w/v (LC2 treatment), were found to be the most effective treatments in enhancing seed germination and tomato growth. In addition, they reduced disease severity by 84.6-97.5%, relative to FORL-inoculated and untreated control, and by 36.2-89.6% compared to pathogen-infected and hymexazol-treated control. FORL mycelial growth on PDA medium amended with aqueous and organic extracts of S. linnaeanum and L. arabicum varied depending on plant organs, the nature of extracts and the tested concentrations. Aqueous, chloroformic, ethyl acetate and butanol extracts were all active against FORL. The most important antifungal activity (expressed as reduction by 41.4-60.8% compared to control) was displayed by leaf butanolic extracts of both species applied at 4% (v/v). High performance liquid chromatography (HPLC) analysis performed for S. linnaeanum butanolic leaf extract revealed the presence of thirteen phenolic compounds including seven phenolic acids, four flavonoids and two stilbenes. The present study is the first one that demonstrates the possibility of valuation of these two wild Solanaceae as sources of fungal biocontrol agents and biomolecules active against FORL with bio-fertilizing and disease-suppressive effects on tomato plants.

Abdellatif, Emna. 2018. Pseudomonas syringae as pathogen of Citrus: Insights into phenotypic and genotypic characterization. Comparative genomics and biological control. Doctorate Thesis in Phytiatry, INAT, University of Carthage, Tunisia, 200 pp. (Public Defense: 14 May 2018)

Pseudomonas syringae pv. syringae (Pss) is the most polyphagous plant bacterium belonging to the P. syringae species which has become recognized as a phylogenetic complex of strains from terrestrial and aquatic habitats. In the light of this and a recent outbreak of so-called Citrus blast and Citrus black pit disease in Tunisia where Pss-like bacteria were constantly isolated, the aim of our study was to investigate if Pss caused this Citrus disease, and if so, to study the disease and its causal agent in depth. The emergence of Citrus blast and black pit in the Tunisian Citrus orchards was observed as early as the spring of 2011. A collection of bacteria with colony morphology similar to type strain of Pss was obtained and characterized. Samples were collected mostly from fields located in the North of Tunisia where disease symptoms were expanding and less from the Center where the disease was virtually absent through 2013-2015.

Based on phenotypic tests (LOPAT and GATTa) and genetic studies (16S rRNA, rep-PCR, PCR-MP) the causal agent was confirmed to be Pss. As more recently Multi Locus Sequence Analysis (MLSA) was described as a reliable technique for species delineation and strain identification in Pseudomonas, we used it for a better species classification in comparison to strains from various countries and hosts. Overall, fingerprinting techniques and housekeeping genes diversity analyses showed that Pss strains from Citrus in Tunisia are homogenous without any geographic distinction and that they are closely related to the LMG5694 Pss strain isolated from Citrus sinensis from Greece in 1962. An eventual diversity was noticed based on the host of isolation like almond. Whole-genome sequences of two Pss strains from Citrus EC33 (from Tunisia) and LMG5496 (from Greece), is providing valuable information on the taxonomic relationships between the Citrus strains and Pseudomonas strains from various hosts. Average nucleotide identity showed a strong correlation with MLSA. Furthermore, in planta pathogenicity testing allowed fulfilling Koch’s postulates and to determine Citrus spp. cultivar susceptibility among the most cultivated ones.

Additionally done, characterization of strains showed that our strains produce the bacterial toxin syringomycin in vitro and they also have the genes syrB and syrD needed for this toxin production. Studies of the population structure using a principal component analyses based on copper sensitivity in vitro and detection of resistance and effector protein genes revealed a remarkable degree of congruence with the previous genetic and genomic clustering.

The detection of a Pss Citrus population resistant to copper during our study led to the search for an alternative sustainable control strategy using epiphytic antagonistic bacteria from the same phyllosphere as the pathogenic ones. Potential antagonistic bacteria belonging to Bacillus sp. and Pseudomonas spp. (P. moraviensis, P. korensis and P. fluorescens) were identified.

Ben Amira, Maroua. 2018. Study of mycoparasitic relationship between Trichoderma harzianum and Fusarium solani in Olive trees: Molecular and functional characterization of aquaporins from Trichoderma harzianum. Doctorate Thesis in Biological Sciences, Faculty of Sciences of Bizerte, University de Carthage, Tunis, Tunisia; Clermont Auvergne University, Clermont Ferrand, France, 96 pages. (Public Defense: 24 May 2018)

Biological disease control through the use of microorganisms has a great potential for future use in integrated pest management. In a multidisciplinary and fundamental context of molecular physio-phytopathology and to provide solutions for the actors in the olive profession and consumers, we have been studying the activity of a fungal biocontrol agent, Trichoderma harzianum (strain Ths97) against the olive tree pathogen Fusarium solani (strain Fso14), which causes major problems for olive production in Tunisia and elsewhere. The study consists of two parts. In the first part, we have demonstrated that Ths97is a biocontrol agent effective against F. solani Fso14 pathogen. Induction of plant defense responses by Ths97 was shown to be partly responsible for the biocontrol effect. In vitro tests further showed that Ths97 develops mycoparasitic activities towards F. solani Fso14, by forming infection structures such as hyphae windings and wedges, appressoria and papillae. In the second part of the study, we investigated the Major Intrinsic Proteins (MIP) superfamily in the Trichoderma genus. This multigenic family has never been investigated in a hyperparasitic fungal species. Seven MIP members are present in T. harzianum and are classified into 3 subgroups: AQP, AQGP and XIP. Their three-dimensional structures and their putative involvement in transport of water and certain polyols have been examined. Finally, their transcription profiles were monitored in Ths97 in planta in antagonistic situations and in vitro in a parasitic situation with Fso14 and show that 4 MIP are expressed and regulated differentially during the interaction. Our work has shown that Ths97 must be considered as a biological control agent and biostimulator of plant defenses, and that MIPs are involved in the trophic relationships between T. harzianum and the environment. These data contribute to the further development of T. harzianum as an efficient biocontrol agent for sustainable protection of olive trees in Tunisia and around the world.

El Hajji, Lobna. 2018. Pathogenicity, identification of Meloidogyne population fungal mycoflora infecting tomato and management method. Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott-Mariem, University of Sousse, Chott-Mariem, Tunisia, 213 pp. (Public Defense: 27 June 2018)

The field investigations carried out in different areas in Tunisia, producing tomato infested by root-knot nematodes revealed the occurrence of three species of Meloidogyne. M. incognita (40%) and M. javanica (37%) were the most prevalent species in different study areas. M. arenaria (6,63%) was found only on three sites; Monastir (12%), Sousse (33,33%) and Kebili (21,43%). The morphological identification of fungi associated with Meloidogyne infestation showed the occurrence of Fusarium genus with an average frequency of 14,2%. The specific diversity showed that F. oxysporum commonly isolated in soil and root matrices with an average frequency of 11,3% on all tomato plots. However, F. solani (6%) was less abundant. The screening of fungal microbiote revealed the prevalence of Aspergillus, Trichoderma and Penicillium genera. The fungal diversity analysis including Shannon-Wienner's and Simpson index showed that mycoflora varied within sampling seasons and prospection localities. The multi-variate analysis (NMDS and CCA) showed the strict correlation between environmental factors in particular temperature, humidity and the fungal community distribution.

The interaction tests between Meloidogyne javanica and Fusarium oxysporum lycopersici (FORL and FOL), characterized morphologically and by molecular tools, confirmed the synergetic relation between the two pathogens on susceptible and resistant tomato cultivars. The root-knot nematodes infection predisposed resistant host plant to ulterior infection by Fusarium. The simultaneous inoculation by tow pathogens or the nematode inoculated before the fungi caused wilting and browning vascular, symptoms not shown with single inoculation by fungi. The root knot nematode incidence on plant host was greater in concomitant inoculation than sequential one. The co-inoculation of susceptible cultivars by two pathogens increased significantly the Fusarium wilt severity.The interaction mechanism involved several factors as the resistance/susceptibility of plant host, inoculation time of each pathogen, Fusarium race and Meloidogyne virulence.

The disease complex of root-knot nematode-Fusarium evaluated on three tomato cultivars with different resistance levels (Riogrande, Colibri and Firenze) showed differential response between cultivars and synergetic relation occurred independently of resistance degree. After the co-infection by RKN and FOL, the enzymatic activities of peroxidase (POX), catalase (CAT), polyphenol-oxidase (PPO) and superoxide dismutase (SOD) were greater in resistant cultivar than susceptible one. The quantification of proteins content differed with pathogens and resistance degree of tomato cultivars. The co-infection enhanced significantly proteins, total phenol and total sugar contents in roots. At late stage, this co-infection reduced significantly mineral nutrients concentrations (Cu, Zn, Mn, and Fe) in roots of all tested tomato cultivars (susceptible, moderate resistant and resistant). Although, the individual inoculation by root knot nematode increased the Cu concentration on roots and the individual inoculation by FORL enhanced significantly Zn contents on roots compared with healthy control. After cytological sections of tomato roots co-infected by two pathogens, results showed that plant host defense may be started at early stage, 7 days post- inoculation. The defense could be provided either by hypersensitivity reaction or mechanical barrier following suberin and structural protein synthesis. The co-infection by both pathogens promoted the colonization of cortex cells by fungal hyphae which invaded epidermal cells. The comparative qualitative proteomic approach showed the modification of root proteome at early infection stage reflecting induced response of plant host towards separated or associated infection by pathogens.

The fungal diversity richness was explored for researching new biological control candidates.Ten fungi genera tested for their antagonistic activities either against M. javanica and/or F. oxysporum f. sp. lycoperisici. The results demonstrated that five genera (Paecilomyces, Lecanicillium, Penicillium, Pochonia and Trichoderma) exhibited a notable nematicide activity. However, the fungicide potential was limited to Trichoderma and Penicillium. The scanning electronic microscopic observations (SEM) showed that Trichoderma and Lecanicillium could broke the nematode cell wall and penetrate inside egg of M. javanica. Furthermore, the Trichoderma isolates coiled along F. oxysporum mycelium and dissolved cells wall. These isolates were identified by molecular analysis as T. harzianum, T. viride and T. asperellum. These species were tested for nematicide and fungicide effect in vitro and in vivo under controlled conditions. The biocide effect in vitro of filtrate culture of these three species differed between different concentrations. The highest culture filtrate concentrations (50, 75 et 100 %) of Trichoderma species inhibited the mycelium growth of F. oxysporum and egg hatching and caused the considerable mortality of M. javanica after 72 h of exposition. The treatment of tomato plant by three Trichoderma species improved the plant growth and decreased the disease incidence caused by nematode and/or pathogen fungus. The mycelium proteome sequencing of three Trichoderma species revealed that several proteins responsible to their action mode like effect proteins (PR proteins) responsible of stimulation of plant host resistance, stress oxidative enzymes, hydrolytic enzymes and antibiotics. The Trichoderma fungus exhibited the potential of combining several modes of action like competition, antibiosis and myco-parasitism proving its use as promoting biological control candidate.

lhem Guesmi-Mzoughi 2018. Characterization and pathogenicity of nematofauna associated to olive in Tunisian orchards. Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott-Mariem, University of Sousse, Chott-Mariem, Tunisia, 217 pp. (Public Defense: 4 July 2018)

In a new view, studies were conducted to determine the structure and diversity of nematode communities associated to olive trees in Tunisia. Surveys have interested 123 olive orchards, in the mainly known regions for olive culture, located from the North to the Center of Tunisia. In these studies, 17 genus of plant parasitic nematodes were identified which are Meloidogyne, Heterodera, Rotylenchulus, Pratylenchus, Zygotylenchus, Longidorus, Xiphinema, Trichodorus, Criconemoides, Paratylenchus, Helicotylenchus, Rotylenchus, Amplimerlinius, Merlinius, Tylenchorhynchus, Neodolichorhynchus and Tylenchus. Free nematodes associated to olive were Rhabditidae, Cephalobidae (bacterivores), Aphelenchidae, Aphelenchoididae, Filenchus spp. (fungivores), Aporcelaimidae (omnivores) and Mononchidae (predators).

Ecological studies showed that the structure and diversity of nematode communities depends on olive orchard modalities and intensification of soils with the irrigation and the presence of cover crops. The intensification of olive orchards enhanced the multiplication of Meloidogyne and Pratylenchus. Soil physico-chemical properties have influenced the nematode community composition. The abundance of Pratylenchus is positively related to the conductivity, clay and silt contents and exchangeable K. However, the abundance of Meloidogyne is positively related to sand content and negatively related to silt content.

Plant parasitic nematode community structure was variable among the regions. It was more diversified in the Center with the intensification of soils. This diversity was studied with the identification of nematode species by morpho-biometric and molecular analysis. In total, 19 species of plant parasitic nematodes were identified which are Meloidogyne javanica, M. incognita, M. arenaria, Heterodera mediterranea, Pratylenchus oleae, Zygotylenchus guevarai, Helicotylenchus oleae, Rotylenchus goodeyi, R. incultus, R. eximius, Tylenchorhynchus mediterraneus, Longidorus africanus, L. euonymus, L. glycines, Xiphinema conurum, X. italiae, X. meridianum, X. pachtaicum and X. robbinsi. P. oleae is a new species identified and 8 species are identified for the first time on cultivated olive which are Rotylenchus goodeyi, R. incultus, R. eximius, Longidorus euonymus, L. glycines, Xiphinema conurum, X. meridianum and X. robbinsi.

Meloidogyne was prevalent in olive orchards visited. Therefore, a pathogenicity experiment was conducted under controlled conditions to evaluate the reaction of 4 olive cultivars, the most cultivated in Tunisia, Chemlali, Chetoui, Koroneiki and Arbequina against M. incognita. The inoculation rates tested are 10, 15 and 20 eggs and J2/ml of soil. No disease symptoms on aboveground plant parts were observed with the three inoculation rates. Although, the presence of root galls, the reproduction factor values were less than 1.0. The cultivars tested, except Koroneiki, seem to be resistant to M. incognita.

In order to control the most important and prevalent plant parasitic nematodes, two experiments were conducted in two olive orchards which one is infested with P. oleae and the other with M. javanica. The biological products tested are Verticillium leptobactrum (60 X 107 de propagules/tree), Vertimec (0,5 ml diluted 2 liters of water/tree) and Novibiotec 7996 (30 ml diluted in 30 liters of water/tree) compared to a non-treated control. These products were not efficient against P. oleae. However, Novibiotec and V. leptobactrum have significantly reduced the population of M. javanica compared to the control.



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