First Report of Colletotrichum gloeosporioides and Colletotrichum siamense, Causing Anthracnose Disease on coffee trees, in Saudi Arabia.

Coffee (Coffea arabica L.) is a promising agricultural commodity in many countries including Saudi Arabia, but crop production is often constrained by diseases. In December 2021, coffee trees had symptoms of anthracnose disease (CAD) were observed in Jazan Province, Saudi Arabia (17°19'00.8"N 43°11'26.8"E), and the incidence was 55%. Affected trees showed dieback and leaves necrosis. On green and ripening berries, slightly sunken and dark brown lesions were occurred; the berries finally become mummified (Fig. S1). For pathogen isolation, symptomatic tissues (4×4mm) of 30 diseased branches and berries samples were surface-sterilized in 1% sodium hypochlorite for 2 min, followed by 70% ethanol for 20 s, rinsed in sterile distilled water and placed on potato dextrose agar (PDA). Cultures were incubated at 26℃ for 8 days in the dark. Eighteen isolates were recovered, and 2 representative single spore isolates (KSU-CgM17, KSU-CsM42) were used for further study. PDA culture of KSU-CgM17 had aerial white mycelium at first and later became gray to grayish black; light salmon to orange conidial masses were observed on the mycelium plate surface as the cultures aged (Fig. S2). Colony produced by KSU-CsM42 was off-white to gray with cottony mycelia and grayish-white on the undersides of the culture after 10 days at 28° (Fig. S2). Conidial shape of these two isolates were both aseptate, cylindrical to nearly straight, hyaline, rounded at both ends. Conidia (n = 50) measurements were 16 to 18.0 µm long × 4.8 to 6.4 µm wide for KSU-CgM17 and 12.6 to 17.5 µm long × 3.2 to 4.5 µm wide for KSU-CsM42. The microscopic and culture features fitted those for Colletotrichum gloeosporioides species complex (Weir et al. 2012). To further identify these isolates, four genomic DNA loci including the partial ITS rDNA region, and CAL, TUB2, and GAPDH genes were amplified and sequenced (Hu et al., 2015). All sequences were deposited into GenBank under accession numbers: OQ791412 & OQ791413 (ITS), OQ786847 & OQ786851 (CAL), OQ786849 & OQ786850 (TUB2), and OQ786848 & OQ786852 (GAPDH) for KSU-CgM17and KSU-CsM42, respectively (Tables S1& S2). A BLAST search of GenBank showed that these pathogens were identified as C. gloeosporioides (KSU-CgM17) and C. siamense (KSU-CsM42). The pathogenicity was tested on detached coffee leaves or green and red berries (Coa et al., 2019). For inoculation, healthy leaves and berries were wounded with a sterilized needle, placed inside petri dishes containing moist filter paper, and then inoculated with a 10-µl droplet of conidial suspension (106 spores/ ml). Sterile distilled water was used as a negative control. Six replicates were tested per isolate and the experiment was repeated once. The inoculated materials were incubated at 25°C and 100% relative humidity for 8 days. Necrotic lesions developed on 100% of the inoculated coffee materials 6 days later, whereas the negative controls were asymptomatic (Fig. S2). Koch's postulates were fulfilled when typical colonies of these species were successfully re-isolated from the from symptomatic tissues. These pathogens were reported previously to affect coffee in Vietnam (Nguyen et al., 2010), China (Cao et al., 2019), and Puerto Rico (Serrato-Diaz et al., 2020). To our knowledge, this is the first record of C. gloeosporioides and C. siamense causing CAD in Saudi Arabia. Further studies on the epidemiology of CAD on arabica coffee plantations as well as effective strategies for managing this disease are needed.

First Report of Basal Rot of Onion Caused by Fusarium oxysporum f. sp. cepae in Saudi Arabia.

Onion (Allium cepa L.) is a globally important crop worldwide including Saudi Arabia. In November 2020, 2-month-old onion plants (cv. Redwing) in commercial fields within the Sajir area of Riyadh region (∼ 1.4 ha), showed symptoms of yellowing, wilting, stunting, bulb discoloration, rot in the basal parts of bulb and decrease in roots. In the advanced stages, the affected plants collapsed and died. The incidence of symptomatic plants ranged from 30 to 65% in the surveyed fields. To isolate the pathogen, symptomatic onion plants (n =20) were sampled. Diseased tissues from roots and bulbs were cut into small pieces (4 × 4 mm), sanitized with 1% sodium hypochlorite solution for 2 min, submerged in 70% alcohol for 20 s, then rinsed with sterile water, before plating on potato dextrose agar (PDA) medium. The plates were incubated at 25°C for 6 days. Subcultures of the mycelia grown out of the diseased tissues produced purplish pink fungal colonies on PDA. On carnation leaf agar, cultures were characteristic of Fusarium oxysporum as described by Leslie and Summerell (2006), with the presence of unicellular microconidia (3.8 to 7.8 × 1.7 to 2.5 µm, n= 50) without septa in false heads or short monophialides and slightly curved macroconidia (16.3 to 28 × 4.2 to 6.1 µm, n= 50) with two to four septa. Older mycelia developed many chlamydospores that were single or in short chains. To further confirm the pathogen identification, DNA was extracted from single-spore cultures of three representative isolates using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (RPB2) DNA were amplified by PCR and sequenced with the following primers: ITS4 and ITS5 (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 2008); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and RPB2 sequences using MEGA7 placed these isolates in the F. oxysporum clade. The ITS, TEF1-α, and RPB2 sequences of an isolate FOC-OR9 were submitted to GenBank (OL721757, OL764494, and OL764495 respectively). To confirm the forma specialis cepae, a fragment of the F. oxysporum f. sp. cepae gene Secreted In Xylem 3 (SIX3) was amplified by PCR (Kalman et al. 2020). The SIX3 amplicon (∼ 277-bp) was sent for sequencing, and the sequence was submitted to GenBank (OL828265). BLASTn analysis of the sequences showed 100% identity with F. oxysporum f. sp. cepae (KP746408). To fulfill Koch's postulates, pathogenicity tests were performed with healthy onion bulbs cv. "Redwing" of 100-150 g each. Prior to inoculation of onion bulbs, the dry bulb scales, one of the fleshy inner scales, as well as the roots were removed. Bulbs were then surface sterilized (as described above) and injected with 20 µl of a conidial suspension (106 spores/ ml) into the basal plate of each bulb and approximately 1 cm deep into the tissue. Six bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 28 °C in the dark. Six bulbs injected with sterile water and six non-inoculated bulbs served as controls. At the 4th week post inoculation, necrotic rot symptoms and brown discoloration were observed on the basal plates of these inoculated bulbs (similar symptoms to those observed in the field), while control treatments showed no symptoms. The pathogen was re-isolated from the basal plates onto PDA and identified morphologically and molecularly as F. oxysporum f. sp. cepae, thus fulfilling Koch's postulates. The test was repeated twice. This pathogen was previously reported causing onion basal rot in United Kingdom (Taylor, et al., 2013). To our knowledge, this is the first report of basal rot in onion caused by F. oxysporum f. sp. cepae in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses for onion growers in Saudi Arabia. Also, Fusarium mycotoxin contamination of onion bulb could pose a public health risk.

Molecular and physiological characterization of Fusarium strains associated with different diseases in date palm.

Several species of Fusarium cause serious diseases in date palm worldwide. In the present work, 14 SSR markers were used to assess the genetic variation of Fusarium strains isolated from diseased trees in Saudi Arabia. We also studied the effect of different temperatures on mycelial growth of these strains. The pathogenicity of four strains of F. proliferatum was also evaluated on local date palm cultivars. Eleven SSR markers amplified a total of 57 scorable alleles from Fusarium strains. Phylogenetic analysis showed that F. proliferatum strains grouped in one clade with 95% bootstrap value. Within F. proliferatum clade, 14 SSR genotypes were identified, 9 of them were singleton. Four out of the five multi-individual SSR genotypes contained strains isolated from more than one location. Most F. solani strains grouped in one clade with 95% bootstrap value. Overall, the SSR markers previously developed for F. verticillioides and F. oxysporum were very useful in assessing the genetic diversity and confirming the identity of Saudi Fusarium strains. The results from the temperature study showed significant differences in mycelial growth of Fusarium strains at different temperatures tested. The highest average radial growth for Fusarium strains was observed at 25°C, irrespective of species. The four F. proliferatum strains showed significant differences in their pathogenicity on date palm cultivars. It is anticipated that the assessment of genetic diversity, effect of temperature on hyphal growth and pathogenicity of potent pathogenic Fusarium strains recovered from date palm-growing locations in Saudi Arabia can help in effectively controlling these pathogens.

Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk.

Antibiotic-resistant Escherichia coli strains including extended-spectrum ß-lactamase (ESBL) isolates are globally widespread in medical, food, and environmental sources. Some of these strains are considered the most pathogenic bacteria in humans. The present work examined the predominance of antibiotic resistance in E. coli strains in wound infections comparing with E. coli strains isolated from a raw milk as a potential source of those strains. The wound infections included abdomen, anus, arm, back, buttock, chest, foot, hand, head, leg, lung, mouth, neck, penis, thigh, toe, and vagina infections. In total, 161 and 153 isolates identified as E. coli were obtained from wound infections and raw milk, respectively. A Vitek 2 system innovated by bioMérieux, France was applied to perform the identification and susceptibility tests. The E. coli isolates that have ability to produce ESBL were detected by an ESBL panel and NO45 card (bioMérieux). Over half of the E. coli were from abdomen, back, and buttock wound infections. More than 50%of the E. coli isolates obtained from wound infections were resistant to cefazolin, ampicillin, cefuroxime, ciprofloxacin, mezlocillin, moxifloxacin, piperacillin, and tetracycline; 70% of the isolates from wound infections and 0% of the isolates from raw milk were E. coli isolates produced ESBL. The data showed that the strains resistance to multi-antibiotic and produced ESBL are more widespread among wound infections than in raw milk.

Correction to: Brevibacillus laterosporus isolated from the digestive tract of honeybees has high antimicrobial activity and promotes growth and productivity of honeybee's colonies.

The original publication of this paper contains a mistake. The correct affiliation no. 3 is shown in this paper.

Brevibacillus laterosporus isolated from the digestive tract of honeybees has high antimicrobial activity and promotes growth and productivity of honeybee's colonies.

The development of novel antimicrobial drugs, as well as the discovery of novel compounds able to promote honeybee's growth, represents major challenges for modern entomology. The main aim of this study was to investigate whether Brevibacillus laterosporus isolated from the digestive tract of Saudi honeybees, Apis mellifera, was able to stimulate colony strength parameters of honeybees and to evaluate its ability to produce antimicrobial agents. Honeybees were collected in Dirab, Riyadh Region, Saudi Arabia, and microorganisms were isolated and identified by 16S ribosomal RNA analysis. Microscopic identification of the microorganism in its native state was facilitated by atomic force microscopy at high-resolution imaging. Active biological compounds were produced by submerged fermentation with B. laterosporus. The fermented broth was subjected to extraction and purification, and then semi-pure compounds were analyzed by gas chromatography-mass spectrometry. The effectiveness of the crude extract and semi-pure compounds as antimicrobial agents was evaluated by susceptibility assays. More than 22% of the microorganisms isolated from the digestive tract of healthy honeybees have been identified as B. laterosporus, this kind of species has a unique shape and morphological structure. The cyclic dipeptide cyclo(Leu-Pro) produced by B. laterosporus showed biological activity against several pathogenic microorganisms. Furthermore, the total counts of workers, closed brood, and open brood, as well as the production of bee pollen and honey, were better in honeybees treated with a B. laterosporus suspension. The data indicated that the B. laterosporus strain isolated from a healthy honeybee might be a novel probiotic and a producer of important biological compounds.

Mitochondrial Molecular Markers for Resistance to Bayoud Disease in Date Palm.

Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis, is a very serious and destructive disease to date palm. Screening of date palm germplasm for resistance to bayoud disease is a crucial step to avoid or alleviate the disease consequences. Fortunately, it was discovered that there are two mitochondrial plasmid-like DNA molecules associated with susceptibility or resistance to bayoud disease. In this chapter, we present a fast, simple, and reliable technique to screen date palm germplasm for the presence of these mitochondrial molecular markers associated with susceptibility or resistance to bayoud.