Identification and distribution of the "Candidatus Liberibacter asiaticus"-Asian citrus psyllid pathosystem in Saudi Arabia.

Citrus greening disease was first reported in Saudi Arabia during the 1970's when characteristic foliar and fruit symptoms were observed in commercial citrus groves, however, "Candidatus Liberibacter asiaticus" (CLas) was not detected in symptomatic trees until 1981-1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the thirteen citrus-growing regions of the country. Citrus trees were surveyed for presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time, quantitative PCR amplification (qPCR) of the CLas ß-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults, revealed trees were positive for CLas detection in ten of the 13 survey regions, however, CLas was undetectable in ACP adults. Phylogenetic and SNPs analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (~1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, USA isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.

Potential of resistance inducers for citrus huanglongbing management via soil application and assessment of induction of pathogenesis-related protein genes.

Huanglongbing (HLB) or citrus greening currently is the most devastating citrus disease worldwide. Unfortunately, no practical cure has been available up to now. This makes the control of HLB as early as possible very important to be conducted. The objective of this study was to investigate the efficacy of the application of salicylic acid (SA) and Phenylacetic acid (PAA) on one-year-old seedlings of different citrus species (Citrus reticulata, C. sinensis, C. aurantifolii) growing on C. volkameriana and C. aurantium by soil drench methods. Factorial analysis of variance showed the percent change in "Candidatus Liberibacter asiaticus" titer and disease severity on a different combination of citrus species growing on the two rootstocks treated with inducers and Oxytetracycline (OTC) were significantly different compared to the untreated plants. SA alone or in combination with OTC provided excellent (P-value < 0.05) control of HLB based on all parameters. The interaction between both factors (Rootstocks x Citrus species) significantly influenced the Ct value (P-value = 0.0001). "Candidatus Liberibacter asiaticus" titer in plants treated with OTC was reduced significantly with a range of -18.75 up to -78.42. Overall, the highest reduction was observed in the application of OTC on sweet orange growing on C. volkameriana (-78.42), while the lowest reduction was observed in the same cultivar which was treated with a combination of SA and OTC (-3.36). Induction of pathogenesis-related (PR) genes, i.e., PR1, PR2, and PR15, biosynthesis of Jasmonic acid and ethylene which are also important pathways to defense activity were also significantly increased in treated plants compared to untreated plants. This study suggests that the application of inducer alone is acceptable for HLB management. We proposed the application of SA and PAA as a soil drench on the citrus seedlings as promising, easy, and environmentally safe for HLB disease control on citrus seedlings.

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.

Characterization of the Asian Citrus Psyllid-'Candidatus Liberibacter Asiaticus' Pathosystem in Saudi Arabia Reveals Two Predominant CLas Lineages and One Asian Citrus Psyllid Vector Haplotype.

In Saudi Arabia (SA), the citrus greening disease is caused by 'Candidatus Liberibacter asiaticus' (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the 'Asiatic' and 'Floridian' strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.

First report of Lasiodiplodia theobromae Causing Dieback, a Destructive Disease on Mango trees in Saudi Arabia.

Mango (Mangifera indica L.) is a popular tropical fruit crop in Saudi Arabia. However, susceptibility to diseases is a major factor that restrict the development of mango trees, reducing the yield and production (Ploetz, 2003). In December 2021, a survey was conducted for mango trees which were showing symptoms of decline in the field located in the district Al-Jumum of Makkah Province, in western Saudi Arabia (21°46'18.9"N 39°35'21.2"E). The disease severity was approximately 40% with 15% incidence of mango trees showing symptoms of twig dieback, leaf necrosis, leaf fall, and internal tissue necrosis as well as darkening within the vascular tissue upon splitting the infected branches. As the disease progressed, the affected branches were turned black-brown and dried up (Supplementary Figure S1). To isolate the pathogen, 20 symptomatic branches were arbitrarily sampled from different parts of the field and washed with tap water. Diseased branches were cut into 4 × 4 mm portions (between symptomatic and healthy tissues), submersed in 70% alcohol for 20 s, surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed with sterile distilled water, and cultured on potato dextrose plates (PDA). The plates were incubated at 25°C in darkness for 3-4 days, and then pure culture of the fungus was obtained by hyphal tip isolation technique. After 3 days of culturing at 25°C on PDA medium, the fungal colonies were grayish-white with uneven edges, and becoming dark grey to black colored after 5 days. After 21 days at 25 ℃ in constant light, the colonies produced dense aerial mycelium at which stage numerous dark colored pycnidia were formed and conidia were observed. Immature conidia were unicellular, hyaline, elliptical or ovate, and truncated at the base, becoming dark brown, thick-walled, one-septate, and longitudinal striation at maturity. Mature conidia measured 22.4±1.6 to 28.7±2.8 µm long and 12.8±1.3 to 15.6±2.4 µm width (n=40). The morphological characteristics of the colonies were consistent with to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (syn. Botryodiplodia theobromae Pat.) (Zambettakis, 1954; Sutton, 1980). Fifteen isolates were obtained, and a single representative isolate (LPT07-KSU) was used for further study. To further confirm the pathogen identification, genomic DNA was extracted from a single-spore culture using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α, (tef1-α) were PCR amplification and sequencing with the following primers: ITS4 and ITS5 (White et al. 1990); and EF-1 and EF-2 (O'Donnell et al. 2008), respectively. The resulting ITS, and TEF1-α, sequences were submitted in GenBank under accession numbers ON192029, and ON209443, respectively. BLASTn analysis of these genes revealed ≥99% identity with the corresponding sequences of L. theobromae in GenBank (MH644067 for ITS region and MZ502303 for tef1-α gene). The result of phylogenetic analysis also showed that the pathogen was identified as L. theobromae, confirming the morphological identification. A pathogenicity assay was carried out on healthy 1-year-old mango cv. "Haden" seedlings. Infection followed the method of Saeed et al., (2017), consisting of excising a 5-mm-diameter tissue bark out of branches (~ 10 to 15-cm of the apical tip) and replacing it with a 5 mm PDA plugs colonized with L. theobromae from 20-days-old-culture or non-colonized plugs (controls). The area of inoculation was covered with parafilm to avoid dehydration. All seedlings were kept under greenhouse conditions (27°C, 16/8-h day/night, 70% RH) and monitored for disease development. Five replicates were used for inoculated and control plants. After 28 days, all inoculated plants displayed similar symptoms to those observed in the field, whereas control plants remained symptomless. Koch's postulates were fulfilled when typical colonies of L. theobromae were successfully re-isolated from the from symptomatic tissues. The test was repeated twice. This pathogen was reported to affect mango cultivation in China (Li et al., 2013), United Arab Emirates (Saeed et al., 2017), and Mexico (Bautista-Cruz et al., 2019). However, to the best of our knowledge, this is the first report of L. theobromae causing dieback disease on mango in Saudi Arabia. The occurrence of manage dieback highlights the importance of disease surveillance in the region. Effective control strategies are need to be established to reduce the losses.

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.

First Report of Fusarium Root and Stem Rot Caused by Fusarium oxysporum f. sp. radicis-cucumerinum on Greenhouse Cucumbers in Saudi Arabia.

Cucumber (Cucumis sativus L.) is an important vegetable crop in Saudi Arabia. During May 2018, 45 - 60% of 5-month-old cucumber plants showed symptoms of a previously unknown wilt in commercial greenhouses around Al Kharj area of Riyadh region. Symptoms consisted of crown and root rot, wilting and stem disintegration, along with yellowish brown to brown external discoloration extended throughout the affected tissues. As the disease progressed, a pinkish-orange mycelial growth was often observed at the basis of affected stems while vessels were discolored. Subsequently, the affected plants were collapsed and died. Crown, stem, and root fragments (4 × 4 mm) were cut from symptomatic tissues, surface sterilized in 2.5% NaOCl, cultured on potato dextrose agar (PDA) with 25 mg/liter of streptomycin sulfate, and incubated at 26°C in darkness for 6 days. Single-spored cultures produced white mycelium with pink, white, or purple pigmentation in the center. The mycelium produced sporodochia. Macroconidia were mainly slightly curved with three to five septa. Microconidia were single-celled oval and produced on short lateral phialides. Chlamydospores were single or in short chains. Morphologically, the isolated fungus was characterized as Fusarium oxysporum (Leslie and Summerell 2006). To further confirm the fungus identification, DNA was extracted from a single-spored culture. 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) with the following primers: ITS4 and ITS5 (White et al. 2017), EF-1 and EF-2 (O'Donnell et al. 2008), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. The ITS, TEF1-α, and rpb2 sequences of the isolate FCKSU17 were submitted to GenBank (MT232918, MW471131, and MW449833 respectively). Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and rpb2 sequences using MEGA7 placed this strain in the F. oxysporum clade. To confirm the forma specialis radicis-cucumerinum, amplification with the specific primers ForcF1/ForcR2 was conducted (Lievens et al. 2007). The amplified fragment (∼ 250-bp) was sent for sequencing, and the sequence was submitted to GenBank (MW471132). BLASTn analysis of the sequences showed 100% identity with F. oxysporum radicis-cucumerinum (KP746408). To fulfill Koch's postulates, pathogenicity test was conducted on 7-day-old plants of cucumber cultivar Beit Alpha grown into pots filled with soil mix (2:1 sandy loam-peat moss, vol/vol). The plants were inoculated through drenching with 100 ml of conidial suspension in sterile distilled water (106 spores/ml) per pot. Control plants were treated with sterile distilled water. Each treatment included 10 replicates (pots), with two plants per pot. The pathogenicity test was repeated once. Cucumber plants inoculated with the fungus showed early wilting symptoms within the first 2 weeks post inoculation. At the 6th week post inoculation, 90 to 100% of the inoculated plants developed typical symptoms. No symptoms were observed on the control plants. The pathogen was successfully re-isolated from the inoculated wilted plants and identified morphologically. To our knowledge, this is the first report of F. oxysporum f.sp. radicis-cucumerinum on cucumber in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses on cucumbers in Saudi Arabia.

Molluscicidal activity of cardiac glycosides isolated from Adenium obesum.

BACKGROUND: Terrestrial mollusks are one of most important agricultural pests worldwide. Natural phytochemicals have an extended history as a source of pesticides. This study was planned to isolate molluscicidal active compounds from the stems of Adenium obesum. RESULTS: The benzene-soluble fraction of the hydroethanolic extract displayed the most potent molluscicidal activity against Monacha obstructa among different solvent fractions with a median lethal dose (LD50 ) of 4.91 µg g-1 body weight (bw). The bioactivity-guided chemical exploration of the benzene-soluble fraction led to the isolation of two known cardiac glycosides, cerberin and neriifolin which showed significant molluscicidal activity with LD50 values of 5.39 and 4.3 µg g-1 bw, respectively. CONCLUSION: Isolation of the cardiac glycoside neriifolin from A. obesum and the molluscicidal activity of cerberin and neriifolin against terrestrial snails are reported for the first time. © 2019 Society of Chemical Industry.

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.

Characterization of Novel Trichoderma asperellum Isolates to Select Effective Biocontrol Agents Against Tomato Fusarium Wilt.

The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL) is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD) method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and ß-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their anta- gonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents.

Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae.

In this study, resistance responses were investigated during the interaction of Botrytis fabae with two faba bean cultivars expressing different levels of resistance against this pathogen, Nubaria (resistant) and Giza 40 (susceptible). Disease severity was assessed on leaves using a rating scale from 1 to 9. Accumulation levels of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzymes (superoxide dismutase, catalase and ascorbate peroxidase) were measured in leaf tissues at different times of infection. The expression profiles of two pathogenesis-related proteins (PRPs) encoded by the genes PR-1 and ß-1,3-glucanase were also investigated using reverse transcription RT-PCR analysis. The accumulation of these defense responses was induced significantly in both cultivars upon infection with B. fabae compared with un-inoculated controls. The resistant cultivar showed weaker necrotic symptom expression, less ROS accumulation, a lower rate of lipid peroxidation and higher activity of the enzymatic ROS scavenging system compared with susceptible cultivar. Interestingly, ROS accumulated rapidly in the resistant leaf tissues and peaked during the early stages of infection, whereas accumulation was stronger and more intense in the susceptible tissues in later stages. Moreover, the response of the resistant cultivar to infection was earlier and stronger, exhibiting high transcript accumulation of the PR genes. These results indicated that the induction of oxidant/antioxidant responses and the accumulation of PRPs are part of the faba bean defense mechanism against the necrotrophic fungus B. fabae with a different intensity and timing of induction, depending on the resistance levels.

Genetic diversity among late blight resistant and susceptible potato genotypes.

RAPD polymerase chain reaction analysis was used to study the genetic diversity among a wild potato variety Solanum demissum (very resistant to late blight) and six potato cultivars (Hanna, Lady-Olympia, Lady-Rosetta, Spunta, Diamant and Cara) varied in their resistance to Phytophthora infestans. Cluster analysis of six potato genotypes showed that, all tested genotypes were separated into two clusters (1 and 2). Cluster 1, included only the wild potato variety (S. demissum), whereas cluster 2 divided into two groups (G1 and G2). Late blight high resistant cultivars Hanna and Cara were grouped in G1. Group 2 included the moderate resistant cultivar Spunta and the susceptible cultivars Diamant, Lady-Rosetta and Lady-Olympia. The potato cultivars that showed highest genetic similarity to the wild potato variety were the resistant cultivars Hanna and Cara. Lowest genetic similarity was obtained with the susceptible cultivars Lady-Rosetta, Diamant and Lady-Olympia. RAPD primer K17 yielded a band with molecular weight of 936 bp found in all susceptible potato cultivars (Lady-Rosetta, Lady-Olympia and Diamant). On the other hand, band with molecular weight of 765 bp were detected in the wild potato and the resistant cultivars Hanna and Cara. Results of this study suggested that, the RAPD marker technique could be beneficial for revealing the genetic variability of different genotypes of potato varied in their resistibility to late blight.