I. H. Al-Mahmooli

Species or Genotypes? Reassessment of Four Recently Described Species of the Ceratocystis Wilt Pathogen, Ceratocystis fimbriata, on Mangifera indica.

Ceratocystis wilt is among the most important diseases on mango (Mangifera indica) in Brazil, Oman, and Pakistan. The causal agent was originally identified in Brazil as Ceratocystis fimbriata, which is considered by some as a complex of many cryptic species, and four new species on mango trees were distinguished from C. fimbriata based on variation in internal transcribed spacer sequences. In the present study, phylogenetic analyses using DNA sequences of mating type genes, TEF-1α, and β-tubulin failed to identify lineages corresponding to the four new species names. Further, mating experiments found that the mango isolates representing the new species were interfertile with each other and a tester strain from sweet potato (Ipomoea batatas), on which the name C. fimbriata is based, and there was little morphological variation among the mango isolates. Microsatellite markers found substantial differentiation among mango isolates at the regional and population levels, but certain microsatellite genotypes were commonly found in multiple populations, suggesting that these genotypes had been disseminated in infected nursery stock. The most common microsatellite genotypes corresponded to the four recently named species (C. manginecans, C. acaciivora, C. mangicola, and C. mangivora), which are considered synonyms of C. fimbriata. This study points to the potential problems of naming new species based on introduced genotypes of a pathogen, the value of an understanding of natural variation within and among populations, and the importance of phenotype in delimiting species.

Population Genetic Analysis Reveals Diversity in Lasiodiplodia Species Infecting Date Palm, Citrus, and Mango in Oman and the UAE

Lasiodiplodia is a common pathogen causing dieback, gummosis, or root necrosis on the three most important fruit crops in Oman and the United Arab Emirates (UAE): date palm (Phoenix dactylifera), Citrus spp., and mango (Mangifera indica). A study was conducted to examine diversity in 64 Lasiodiplodia isolates infecting date palm (24), Citrus (11), and mango (29) in Oman and the UAE. Identification based on sequences of the internal transcribed spacer (ITS) rDNA and EF1α gene showed that date palm isolates belonged to L. hormozganensis (75% of isolates) and L. theobromae (25%); Citrus isolates belonged to L. hormozganensis (45%), L. theobromae (45%), and L. iraniensis (10%); and mango isolates belonged to L. theobromae (59%), L. iraniensis (34%), and L. hormozganensis (7%). Amplified fragment length polymorphism (AFLP) fingerprinting of the 64 isolates using four primer pair combinations produced 64 genotypes and 972 polymorphic alleles. Cluster analysis separated the isolates into four clusters representing the three species. A higher level of genetic diversity was observed in L. iraniensis (0.3105) compared to L. hormozganensis (0.2503) and L. theobromae (0.2331) in Oman. Analysis of molecular variance (AMOVA) indicated the existence of low levels of genetic differentiation among date palm populations of L. hormozganensis obtained from Oman and the UAE (FST = 0.025) and among populations of L. hormozganensis (0.0485) and L. theobromae (0.0703) from date palm, Citrus, and mango. These findings imply a high rate of movement of L. hormozganensis and L. theobromae isolates among date palm, Citrus, and mango and between the two countries. Findings from the pathogenicity test supported the AMOVA analysis and suggested a lack of host specialization in L. hormozganensis, L. iraniensis, and L. theobromae on date palm, acid lime, and mango. Although this is the first record of L. hormozganensis and L. iraniensis in Oman, the relatively moderate level of genetic diversity in the two species compared to L. theobromae suggests that the two species have been in Oman for a long time but misidentified by morphology and ITS rDNA sequences as L. theobromae. This study is also the first record of date palm and acid lime as natural hosts for L. hormozganensis and the first record of L. hormozganensis in the UAE. The diversity in Lasiodiplodia species affecting date palm, Citrus, and mango in Oman and the UAE should be taken into consideration when planning future management programs for diseases caused by these pathogens.

A new disease in Citrus aurantifolia in Oman, “sudden decline”, is associated with a pathogen complex including a 16SrII group phytoplasma

Witches’ broom disease of lime is a devastating disease in the Sultanate of Oman. In recent years a new disease has been observed, which promotes a sudden decline in Citrus aurantifolia (acid lime). The etiological agent of this new disease has not yet been determined. In this study the presence of phytoplasmas and RNAs was evaluated in plants showing the sudden decline symptomatology. The results reveal the presence of a complex of at least three different pathogens: 16SrII phytoplasmas, Citrus tristeza virus and Citrus exocortis viroid.

First Report of Euphorbia larica Dieback Caused by Fusarium brachygibbosum in Oman

Euphorbia larica Boiss. (Arabic = Isbaq) is a dominant and common component of the native desert flora of northern Oman. Traditional ethnobotanical uses have included use of the latex for treating camels with parasites. In February 2011, E. larica plants showing stem lesions up to several cm long and in many cases with stem dieback were collected from Al-Khoudh 50 km west of Muscat. The disease appeared widespread within the location where several dead specimens were also recorded, although the cause was unclear. Sections (5 mm) of five diseased branches taken from different plants and placed on potato dextrose agar (PDA) in all cases yielded Fusarium-like colonies. Colonies recovered were initially white becoming rose to medium red in color with abundant aerial mycelium. Macroconidia were scarce and scattered (mean of 20 spores: 26.83 × 4.73 μm) with three to four septa per spore; microconidia were slightly curved, ovoid, and fusiform (mean of 20 spores: 11.64 × 4.03 μm) with zero to two septa per spore. Spherical chlamydospores (mean of 20 spores: 11.05 μm) were terminal and intercalary, single, and in chains. In vitro characters and spores measurements conformed to previously described features of Fusarium brachygibbosum Padwick (1). Mycelial plugs (5 mm) were taken from 7-day-old cultures of the fungus grown on 2.5% PDA and applied to a small incision (3 mm) on the stems of healthy E. larica grown in situ and protected with wet cotton and Parafilm. The residual agar, mycelium, cotton, and Parafilm were removed after 7 days and symptoms were recorded. Control stems were inoculated using PDA (5 mm) plugs alone and inoculations were repeated twice. Artificial inoculations resulted in dieback of all stems within 11 days and fungal colonies identical to initial isolations were recovered from artificially infected surface-sterilized stem pieces. Identification of F. brachygibbosum was confirmed by comparing sequences generated from the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1 and ITS4 primers) and the intron region of translation elongation factor alpha (EF1-α) (EF-1-986 and EF-728 primers). The ITS and EF1-α sequences were found to share 100% and 99% nucleotide similarity to previously published sequences of the ITS (HQ443206) and EF1-α (JQ429370) regions of F. brachygibbosum in GenBank. The accession number of ITS sequence of one isolate assigned to EMBL-Bank was HF562936. The EF sequence was assigned to EMBL-Bank accession (submission number Hx2000027017; number will be sent later). This pathogen has previously been reported on date palm (2) in Oman but, to our knowledge, this is the first report of this pathogen on E. larica. References: (1) A. M. Al-Sadi et al. Crop Prot. 37:1, 2012. (2) G. W. Padwick. Mycol. Pap. 12:11, 1945.

Development of Resistance to Hymexazol Among Pythium Species in Cucumber Greenhouses in Oman

A study was conducted to characterize the common Pythium spp. in greenhouses in Oman and their level of resistance to hymexazol, a widely used fungicide in the country. Pythium isolates were obtained from soil samples, cocopeat bags, and cucumber roots collected from seven regions in the country. Identification of 80 Pythium isolates to the species level using sequences of the internal transcribed spacer region of the ribosomal RNA showed that they belong to four species: Pythium aphanidermatum (77 isolates), P. spinosum (1 isolate), P. myriotylum (1 isolate), and P. catenulatum (1 isolate). Investigating the aggressiveness of three Pythium spp. on cucumber showed that P. aphanidermatum, P. myriotylum, and P. spinosum are pathogenic. Phylogenetic analysis of P. aphanidermatum isolates showed that most of the isolates obtained from cocopeat clustered separately from isolates obtained from soil and roots. This may indicate a difference in the origin of the cocopeat isolates. Evaluating the resistance of 27 P. aphanidermatum isolates to hymexazol showed that most isolates were sensitive (0.9 to 31.2 mg liter-1) whereas one isolate was resistant (142.9 mg liter-1). This study is the first to report P. myriotylum and P. catenulatum in Oman. It is also the first to report the development of resistance to hymexazol among P. aphanidermatum populations from greenhouses. Growers should use integrated disease management strategies to avoid further development of resistance to hymexazol.

First report of root rot and crown necrosis caused by Pythium aphanidermatum on Phaseolus vulgaris in Oman.

In March 2013, 90% of mature bean plants (Phaseolus vulgaris L. cv. Kendo) grown on a commercial farm in the north of Oman (Barka) developed symptoms of root rot and necrotic streaks on the crown area of the stem and wilted. A Pythium spp. was isolated consistently from roots and basal stems on 2.5% potato dextrose agar (PDA) and V8 (100% vegetable juice) plus 1.5% agar technical. Colonies of Pythium spp. on PDA and V8 plus agar developed abundant aerial mycelia, with the main hyphae being up to 10 μm wide. Zoosporangia were made up of terminal complexes of swollen hyphal branches of different lengths and up to 22 μm wide. Oogonia were terminal, globose, and smooth with a 26-μm diameter (average of 20). Antheridia were mostly intercalary, sometimes terminal, and broadly sac-shaped, 15 μm long and 11 μm wide (average of 20). Oospores were aplerotic, 23 μm in diameter (average of 24), with walls 1 to 2 μm thick at 25°C (ambient temperature). The internal transcribed spacer of the ribosomal DNA (ITS1 and ITS4) sequence of the isolates matched the sequence of Pythium aphanidermatum (Edson) Fitzp. in GenBank. The sequence of isolate Py1 was deposited in GenBank as Accession No. KM102739. This isolate was identified as P. aphanidermatum on the basis of morphological and cultural characteristics (1) and the ITS rDNA sequence. The ITS was found to share 100% nucleotide similarity to previously published sequences of the ITS (KJ755088). To fulfill Kochs postulate, a 5-mm plug of 5-day-old mycelium of isolate Py1 grown on 2.5% PDA was used to inoculate healthy seedlings of beans cv. Kendo. The plug was placed adjacent to the bean stem; PDA served as a control. Five replicate plants were used for the treatment and the control. The plants were maintained in a glasshouse at a temperature of 23 to 25°C. The plants were watered every day. The irrigation water had an electrical conductivity value of 0.2 dSm-1. Eleven days after inoculation, 90% of the plants developed root rot, crown necrosis, and wilt symptoms similar to those observed in the field. On the other hand, control plants did not show any symptoms. The pathogen was re-isolated from roots and basal stems of symptomatic plants. To our knowledge, this is the first report of P. aphanidermatum as the causal agent of root and crown necrosis of mature bean plants in Oman. Future studies should focus on evaluating management options for this disease to avoid possible losses in a crop that has a high export value in Oman. Reference: (1) Y. Serrano et al. Plant Dis. 92:174, 2008.

Expression of phytoplasma-induced witches' broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia L.) in Oman and UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Ca. P. aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semi-tropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II-B. Quantitative real time PCR analysis showed that the concentration of phytoplasma is significantly higher (8,800-801,000 copies) in leaves developing WBD symptoms compared to 2-268 copies in asymptomatic leaves from the same trees and 8 to 874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavorable environmental conditions. These findings could provide a basis for managing witches’ broom disease through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression. This article is protected by copyright. All rights reserved.

Differential expression and phytohormone unbalance in Citrus aurantifolia plants during “sudden decline of lime”, a new phytoplasma disease of citrus

For decades, the production of acid lime (Citrus aurantifolia) in Oman has been affected by diseases caused by phytoplasmas, notably witches’ broom disease of lime (WBDL). In recent years, a new phytoplasma strain in Oman has been observed that promotes the sudden decline of lime (SDL). The molecular mechanisms behind its pathogenicity and mode of interaction with citrus host plants is still completely unknown. In this study, we evaluated the differential expression of genes in symptomatic and asymptomatic lime trees in Oman using a real-time quantitative PCR assay. Among 27 regulatory and biosynthesis-related genes tested in Citrus aurantifolia plants during phytoplasma infection, we verified the presence of 14 responsive genes in plants showing SDL symptoms, revealing a specific set of SDL-responsive genes. Quantification data of endogenous 3-indoleacetic acid and jasmonic acid show an unbalanced hormonal content in symptomatic lime trees, corroborating the gene expression data. The identification of regulatory genes differentially expressed in plant-phytoplasma interactions during SDL will help to elucidate the mechanisms possibly involved in defense responses, development and death-triggered signals in infected citrus plants.

Genetic analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom on acid lime trees

“Candidatus Phytoplasma aurantifolia” is associated with witches’ broom disease of lime in Oman and the UAE. A previous study showed that an infection by phytoplasma may not necessarily result in the physical appearance of witches’ broom symptoms in some locations in Oman and the UAE. This study investigated whether phytoplasma strains belonging to “Ca. P. aurantifolia” (based on the 16S rRNA gene analysis) in locations where disease symptoms are expressed are different from phytoplasma in locations where disease symptoms are not expressed. About 21 phytoplasma strains (15 from areas and trees with disease symptoms and six from areas and trees without disease symptoms) were included in the analysis. The study utilized sequences of the imp and SAP11 genes to characterize the 21 strains. Phylogenetic analysis of both genes showed that the 21 strains are similar to each other and to reference strains in GenBank. The study shows that there is a low level of diversity among all phytoplasma strains. In addition, it shows that phytoplasma in places where witches’ broom symptoms are not expressed are similar to phytoplasma in places where disease symptoms are expressed. This may suggest that disease expression is not linked to the presence of different phytoplasma strains, but may be due to other factors such as weather conditions.

Identification of Mungbean yellow mosaic India virus infecting cucumber in Oman

Cucumber (Cucumis sativus) is a widely cultivated plant in the family Cucurbitaceae that is grown as greenhouse crop in Oman. During a recent survey for begomoviruses, whitefly infestation was observed in two greenhouses of cucumber in the Barka region. Plants were exhibiting a foliar yellow mosaic and crumpling, symptoms typical of begomovirus infection (Supplementary Figure 1), with an incidence of 50-60 %. Leaves from four symptomatic plants were collected, two plants from each greenhouse, as well as leaves from two apparently healthy (symptomless) plants, one from each greenhouse. Electron microscopy analysis of negatively stained sap samples showed the presence of geminate (twinned quasi-icosahedral) particles of ~18-20x30 nm in samples from symptomatic but not non-symptomatic plants, suggesting the presence of a geminivirus. Nucleic acids were extracted from leaf samples (Porebski and Bailey 1997) and used in PCR with primers to the conserved domain of the coat protein of begomoviruses (Brown et al....