First Report of Leaf Wilt Caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia

Abstract

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in ‘Deglet Nour’ trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O’Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned ‘Deglet Nour’ plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch’s postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.

Pest categorisation of Fusarium brachygibbosum

“…-Allium cepa (Tirado-Ramirez et al, 2021); -Beta vulgaris (Cao et al, 2018); -Cannabis sativa (Punja et al, 2018(Punja et al, , 2019; -Citrullus lanatus (Renter ıa-Mart ınez et al, 2015); -Citrus spp. (Al-Sadi et al, 2014); -Glycine max (Wang et al, 2021); -Nerium oleander (Mirhosseini et al, 2014); -Nicotiana tabacum (Qiu et al, 2021) -Phoenix dactylifera (Al-Sadi et al, 2012;Saleh et al, 2017;Al-Nadabi et al, 2020;Namsi et al, 2020;Nishad and Ahmed, 2020;Rabaaoui et al, 2021); -Prunus dulcis (Seidle, 2016;Stack et al, 2017); -Solanum tuberosum (Zimudzi et al, 2017;Azil et al, 2021); -Sorghum vulgare (Padwick, 1945); -Trifolium subterraneum (Tan et al 2011); -Triticum spp. (Ali et al, 2020;€ Ozer et al, 2020;Mohammed-Ameen et al, 2021;Van Coller et al, 2013); -Vitis vinifera (Akg € ul and Ahio glu, 2019); -Zea mays (Shan et al, 2017;Fallahi et al, 2019).…”

Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia

“…In this study, we report for the first time an extensive study on the Alternaria species occurring on date palm leaves showing leaf spot symptoms, in Tunisia. In recent years, a high incidence of leaf spot disease was recorded on date palm ( Namsi et al, 2021 ). In the period 2017–2019, we inspected palm date plantations located in Djerib, the most intense area of date production in Tunisia.…”

“…In addition to the above mentioned species, other species have been associated with leaf blight in Tunisia. Ben Chobba et al (2013) identified also A. gaisen (included in Alternaria Section) by using ITS1 and ITS4 genes, whereas Namsi et al (2021) identified A. mali , by using ITS , gpd , CaM , and Alt-a1 genes. However, in other studies, A. mali was proposed as synonymous to A. alternata species ( Woudenberg et al, 2015 ), whereas, according to Somma et al (2019) , A. mali was phylogenetically more similar to A. gaisen , both species being distinguishable from A. alternata .…”

“…In particular, Alternaria alternata is the most important Alternaria species detected in all date palm cultivated areas. However, other Alternaria species also have been reported from symptomatic date palm leaves, such as A. burnsii and A. arborescens , in Oman ( Al-Nadabi et al, 2018 ); A. chlamydospora and A. radicina in Iraq ( Abass et al, 2007 ; Khudhair et al, 2015 ); A. bokurai , A. arborescens and A. mali in Tunisia ( Ben Chobba et al, 2013 ; Namsi et al, 2021 ). Most of these reports were based only on morphological identification.…”

Phylogeny and Mycotoxin Profile of Pathogenic Fusarium Species Isolated from Sudden Decline Syndrome and Leaf Wilt Symptoms on Date Palms (Phoenix dactylifera) in Tunisia

“…The strains were grown for 7–10 days at 25 °C on PDA under a light/darkness photoperiod of 12 h. For each strain, the inoculum was prepared by flooding the agar plate surface with 10 mL of sterile distilled water (SDW) and scraping with a spatula. Conidial suspensions were filtered through four layers of cheese cloth and were diluted with SDW for obtaining a final concentration of 10 5 conidia mL −1 [ 64 ].…”