Fusarium equiseti as one of the main Fusarium species causing wilt and root rot of chickpeas in Morocco

Fungal isolates of Fusarium were collected from symptomatic chickpea ( Cicer arietinum L.) plants growing in fields within Souk Tlat commune in the Gharb region. Morphological and molecular characterizations were performed of the fungal isolate N3 obtained from a chickpea plant. PCR amplification and sequencing of the internal transcribed spacer using the primers ITS1 and ITS4 was applied to identify the fungal isolate N3. The maximum similarity index of the fungus was found to be 99.33% with Fusarium equiseti (accession no. MT111122). In the pathogenicity test, both chickpea seed dip inoculation and soil infestation by the spore suspension of Fusarium isolate were adopted. Four weeks after chickpea seed inoculation, few plants emerged and those that emerged were stunted


Introduction
Chickpea (Cicer arietinum L.) is one of the oldest cultivated protein legumes in the world. It is mainly used for human consumption and is an essential constituent of the Mediterranean diet and basic food in Pakistan and India (Millan et al., 2010). Domesticated in association with other crops such as wheat and barley, Cicer arietinum L. is believed to be a part of the agricultural revolution, and in terms of consumption, ranks second after broad bean (Gupta et al., 2014).
In Morocco, the cultivated chickpea area covers an acreage of 54000 ha (MAPMDREF, 2020a), with a production of 49700 T recorded in the 2019/2020 agricultural season (MAPMDREF, 2020b). Most of the agricultural systems of chickpea production in the country suffer from several constraints, mainly biotic and abiotic factors that cause serious damage before and after harvest.
The widespread high temperature and drought stress in different regions of chickpea production can affect flowering and pod setting stages which lead to decreases in chickpea yield (Houasli et al., 2020). Previous reports on the chickpea crop have recorded the presence of Ascochyta rabiei, the causal agent of blight disease in Morocco, in all chickpea areas (Grewal, 1984;Singh, 1984) Bencheqroun et al. (2022) pointed out that Didymella rabiei (Kovatsch.) Arx. is the most devastating fungal infection of chickpea crops inflicting considerable yield and quality losses.
Isolates of Fusarium and F. solani were recovered from necrotic lesions of chickpea roots in different chickpea growing areas (El Hazzat et al., 2019). Nevertheless, taxonomic confirmation of which species of Fusarium causes this necrosis is lacking because numerous species are important plant pathogens (Austwick, 1982). Additionally, the differentiation of Fusarium species through morphological characters is imprecise; hence, the use of molecular techniques become more efficient and accurate for the discrimination of fungal species (Steenkamp et al., 2000). Therefore, the present study was carried out to identify an isolate of Fusarium sp. collected for the first time in chickpea fields in Morocco from diseased chickpea plants based on morphological characters and molecular and pathological characterization via fulfillment of Koch's postulates.

Fungal material
Fusarium isolates was obtained from necrotic lesions associated with infected stem samples of chickpea plants that were grown in different fields in Souk Tlat in Gharb Province of the Rabat-Sale-Kenitra region, Morocco. One hundred plants were chosen at random from the fields. One stem base sample from each plant was analyzed.
Pieces of diseased tissues were rapidly disinfected with 90% alcohol for 5 min, rinsed three times with sterile distilled water, and dried with sterile filter paper. Samples were then placed onto potato dextrose agar plates (BIOKAR Diagnostics) and incubated at 25 °C for 7 days. The colonies formed were transferred to potato sucrose agar (PSA) medium containing potato, sucrose, agaragar, and distilled water. Agar plates were incubated in the same conditions and then observed for species determination. The Fusarium sp. isolate was cultivated in Petri dishes containing PSA medium. The medium was poured into Petri dishes containing 100 mg/L of chloramphenicol at a rate of 30 to 40 mL per dish. Incubation of cultures was performed in the dark at 25 °C for 7 days followed by macroscopic and microscopic characterization depending on the age of the cultures.

Morphological characterization
Macroscopic examination of Fusarium sp. was carried out according to the development of the presence of the pinkish color of the colony, as well as growth and spore production. The microscopic characteristics of the Fusarium N3 isolate were determined under an optical microscopic to confirm the species identity of this pathogen.

Molecular analysis and identification
Molecular identification of the Fusarium N3 isolate was performed after 5 days of culture on PSA medium. DNA extraction was performed according to the method described by Murray and Thompson (1980) and Doyle et al. (1987). DNA amplification of the internal transcribed spacer (ITS) rDNA region was performed using polymerase chain reaction (PCR) using universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990). The PCR reaction was carried out in a reaction mixture of 25 μL containing 5 µL of 5× buffer (MyTaq Reaction Buffer, Bioline, London, UK,), 1 µL of each primer (10 µM), 0.2 µL of MyTaq DNA polymerase (Bioline, London, UK) (5 U µL -1 ), 1-2 μL of template DNA (100 ng) and Milli-Q water to complete the volume. A Veriti thermal cycler (Applied Biosystems) was used for the PCR with the following conditions: initial denaturation at 95 °C for 1 min; 35 cycles of denaturation at 95 °C for 15 s, annealing at 52 °C for 20 s, and extension at 72 °C for 15 s; and a final elongation of 72 °C for 3 min. The quality of the PCR products was verified by electrophoresis on 1% agarose gel in the presence of a 100 bp molecular weight marker.
Sequencing was performed using an ABI PRISM BigDye Terminator v.3.1 Ready Reaction Cycle Sequencing Kit and primer set ITS1 and ITS4. The sequencing products were run on an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems) using the POP-7 polymer. The sequence resulting from this study was submitted to GenBank under the accession no. MT111122, The obtained ITS sequence was then compared with the homologous nucleotides sequence in the GenBank database using the Basic Local Alignment Search Tool (BLAST)
The seeds of a local variety of chickpea intended for pathogenicity testing were surface sterilized by soaking for 5 min in a 10% NaOCl solution, rinsed in sterile distilled water, and then dried on filter paper. The pathogenicity of the N3 isolate was checked through two inoculation techniques.
Technique 1: the surface-disinfected chickpea seeds were inoculated by soaking in water containing a conidial suspension of 10 6 spores/mL of the Fusarium isolate N3 at room temperature (20 °C) for 1 h. The control seeds were treated with only sterile distilled water. The inoculated and control chickpea seeds were sown in plastic pots (13 cm × 13.5 cm) containing autoclaved sieved Mamora forest soil at the rate of 5 seeds/pot. The Mamora forest soil used is loose, very sandy, and slightly basic pH (7.27), with an organic carbon content of 0.35% to 0.6% (Mouria, 2009).
The soil was sieved and sterilized three times at an interval of 24 h at 200 °C for 2 h, and then distributed in the plastic pots (13 cm × 13.5 cm) at the rate of 2 kg of soil per pot.
Technique 2: The culture substrate was inoculated by pouring 15 mL of the conidial suspension of the N3 isolate (Fusarium) at a concentration of 10 6 spores/mL into each pot containing sterile soil. The fungus was allowed to settle for 48 h in the growing medium. The previously disinfected chickpea seeds were sown into these pots at the rate of 5 seeds per pot. Three replicates were prepared for each of the treatments (each pot was a replicate). Two lots of chickpea cultivation pots (control and inoculated) were brought back to the greenhouse to promote seed germination, plant growth, and symptom development monitoring.
After 4 weeks, plant emergence and plant survival were determined in pots containing inoculated seeds. In the pots with inoculated culture substrate according to the second inoculation technique, plant emergence and disease symptoms on chickpea plants were noted after 8 weeks, followed by

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Vol. 57, Article 576 DOI: 10.5586/am.576 assessment of disease severity by calculating the leaf damage index according to the scale established by Douira and Lahlou (1989).
The scores related to the number of leaves constitute the foliar alteration index, calculated according to the formula below (Douira & Lahlou, 1989): At the end of the trial, the pots were brought back to the laboratory to re-isolate the pathogen from the different parts of the plants (roots, crown, stems, and leaf petioles) obtained either from the inoculated seeds (technique 1) or inoculated growing substrate (technique 2). The different parts were separated and disinfected with 95% alcohol for 2 min. Samples were then rinsed several times with sterile distilled water, dried quickly on sterile filter paper, transferred to PSA medium, and incubated in the dark at 25 °C. The microscopic observation was performed after 1 week.

Notes
Appearance of leaves 0 Healthy appearance True leaf: necrosis 5 True leaf: fall The re-isolation percentage (RP%) was calculated using the following formula: RP = (Ns PX /NT) × 100, where Ns PX is the number of segments containing the fungal species X and Nt is the total number of segments used for re-isolation.
After 8 weeks, the lengths of the aerial and root parts of the chickpea plant were measured with a double decimeter and the number of leaves and pods of each plant was counted. The fresh weights of the aerial and root parts were recorded using a precision balance and the dry weight of these parts was recorded after being dried in the oven at 70 °C for 48 h.

Results
The isolate N3 of Fusarium equiseti was obtained from symptomatic samples among a complex of fusarium species including F. solani and F. oxysporum. The percentage of isolation was 3% in the case of F. equiseti.
On the PSA medium, the isolate N3 developed a colony with an abundant aerial mycelium which was fluffy and beige-white colored (Figure 1). Under a microscope, the mycelial filaments were septate. Chlamydospores were present (7 to 13 μm in diameter, spherical, globular, most often intercalary, solitary,or in pairs, and frequently as short chains). Macroconidia were numerous, slightly curved, usually with 5 to 6 septa and 31 to 45 μm long. The description was identical to that of F. equiseti (Corda) Saccardo, reported by Leslie and Summerell (2006)   Plants which were grown in the culture substrate inoculated with F. equiseti also presented a lower root length, fresh and dry weight of root or aerial parts attaining 19.73 cm, 5.33 and 4.12 g, and 5.47 and 4.02 g, respectively, than plants grown in uninoculated soil (28.33 cm, 7.33 and 6.60 g, and 6.66 and 5.40 g, respectively).
The re-isolation performed on the plants grown either from inoculated seeds or on soil infested with F. equiseti confirmed the presence of the fungus in different parts of the plants. Using the

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Vol. 57, Article 576 DOI: 10.5586/am.576 first technique, the highest percentage of the pathogen re-isolation was registered in the chickpea root with 70.22% followed by collar (70%), stem (60%), and leaf petiole (34.33 %), whereas percentages recorded in chickpea plants after soil inoculation were 84.77% (collar), 72.77% (root), 64.44% (stem), and 36.11% (petiole). Fusarium equiseti was consistently reisolated from infected seeds and collar tissues, satisfying Koch's postulates. To our knowledge, this is the first report of Fusarium equiseti causing seed rot and discoloration at the level of the crown and wilting of chickpea plants.

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Fusarium equiseti has also been isolated from cereals such as corn, wheat, and barley (Ballois, 2012). This fungal species is responsible for rotting stems and premature wilting of corn plants (Swamy et al., 2020) and root rot in winter wheat (Booth 1971). Sometimes, this pathogen is associated with blight of wheat ears (Shaner, 2003;Gale, 2003;Tekauz et al., 2005;Wing et al., 1993;Xue et al., 2006) and rice panicles. Infection can also occur during grain storage or afterward (Hashem et al., 2010). In Morocco, several Fusarium species are associated with symptoms of root rot in cereals; the most common are F. equiseti, F. culmorum, F. oxysporum, and F. solani (Lyamani 1988). effective control program, Fusarium equiseti will probably become an important pathogen and take its place among the other known diseases of chickpea.

Conclusion
To our knowledge, the present study showed for the first time that Fusarium equiseti can play serious role in the disease process of chickpea in Morocco causing seed rot, root and and crown necrosis and leaf yellowing.