Characterization, Phytopathogenicity and Host Range Studies of Neoscytalidium Dimidiatuml. (Botryosphaeraceae) Associated With Dieback of Lebbeck Trees (Albizia Lebbeck L. Benth) in Saudi Arabia


 A survey in 2016 showed that more than 80% lebbeck trees inside the main campus of Qassim University were wilted and dead. Symptoms of dieback, root rot, stem cankers and decline were observed in the trees. The trunks exhibited black masses of spores which soon spread to other, healthy trees. A fungus, having arthroconidial and asexual synanamorph characteristics, and was identified as Neoscytalidiumdimidiatum KSA of the class Coelomycetes within the family Botryosphaeraceae and was subsequently isolated from the infected lebbeck trees. Six-year-old lebbeck seedlings were inoculated with the N. dimidiatum KSA isolate. Symptoms of chloroses followed by dropping leaves appeared four weeks after inoculation. The fungus re-isolated from the infected seedlings expressed the same morphological characteristics on the culture media as the N. dimidiatum KSA isolate. A host range study involving six different tree species were inoculated under growth chamber conditions using the identified isolate of the N. dimidiatum KSA fungus. Four weeks after the inoculation, three of these species exhibited wilting and died. To the best of our knowledge, this study is the first to report on N. dimidiatum in Saudi Arabia.


Introduction
The genus Albiziaincludes about one hundred and fty species, mostly trees and shrubs native tothe tropical and subtropical regionsof Asia and Africa{23}.Albizia lebbeck is partiallyidenti edwith deciduous and semi-deciduous forestswithin Asia from eastern Pakistan through India to Sri Lanka and Myanmar.
The tree has been introduced as an ornamental and plantation tree throughout tropicaland northern subtropical regions which includeCentral America, Colombia, Venezuela and Brazil. {13}.
Albizia lebbeckis a fast-growing, medium-sized deciduous tree with nitrogen xation and assimilation capabilities. The treehasan umbrella-shaped crown of thin foliage and a smooth, nely ssured trunk withgreyish-brown bark. Depending on site conditions, annual crown cover increases can spread from 0.5 to 2.0 m.Individual trees attain an average maximum height of18 -25 m and 50 -80cm d.b.h.The species grows well at altitudes up to 1500 m a.s.l. in sites receiving 500 -2500 mm annual rainfall and it can tolerate both light forest and drought conditions. Although it grows poorly in heavy clay soils, it tolerates saline, sodic or lateritic soils. The tree developswell in moist,well-drained soil. Its leaves, seeds, Many years ago, A. lebbeck was introduced to central Saudi Arabiafrom India and it has adapted well to the severe environmental conditions {14}.The tree is characterized by its resistance to heat and drought andtolerance of harsh environments.
The Botryosphaeriaceaeinclude various morphologically diverse fungi that act as pathogens, endophytes or saprobes, mainly on woody hosts. They are found in all geographical and climatic areas of the world, with the exception of the polar regions. Their persistent association with plant diseases has stimulated substantial interest in these fungi, much of which has been focused on the systematics of species and genera{27}.
Neoscytalidium hyalinum is a plant fungus belonging to the family Botryosphaeriaceae. Nattrass{24} rst speci edit by the name Hendersonula toruloidea.In 1970, Gentles  The aim of this study was to identify and characterize the Botryosphaeriaceae occurring on A.lebbeck inSaudi Arabia using themorphology of the anamorph stages,PCR-RFLP analysis, DNA sequence comparisons, host range studies and the pathogenicity of these fungi.

Symptomatology, sample collection and isolation
The branches and main stems of A. lebbeckexhibitedextensive dieback, decline and cracking as the main symptoms of the samplescollected throughout the surveyconducted on the main campus of Qassim University in Buraydah in central Saudi Arabia. The dieback symptomson the lebbeck trees were predominant over 80% of thesurveyed area. Small specimens (4 -5 mm) from the bark of the main stems and live tissue from the brancheswere collectedin polythene bags and transported to the phytopathology laboratory of theQassim University College of Agriculture and VeterinaryMedicine for initial fungal isolation and further analysis. In addition, pure cultures of the fungus were sent to the Leibniz Institute DSMZ -German CollectionofMicroorganisms and Cell Cultures, Braunschweig, Germany, for molecular analysis and deposition. Thesurfacesof thespecimenswere disinfected for 1 min in 70% ethanol followed by3 min in 1% sodium hypochloride andthereafter washed thoroughly two times withrenewedsterile distilled water and dried on sterile tissues.Using sterile forceps, the pieces were placed onto sterilized disposable petri dishescontainingautoclaved potato dextrose agar (PDA, Oxoid, UK) to which was added 0.5 g/L streptomycin sulphideand thenincubatedat ± 25°Cfor 72 h.

Morphological analysis
The isolate was then placed in petri dishes containing autoclaved 2% water agar (WA Agar, Oxoid, England)using a sterile casuarina needle and incubated for three weeks at ± 25°Cto encourage sporulation of the culturesfor conidial characteristics. In order to identify the species, the variance of morphological characteristics were detected using a light microscope (OLYMPUS CX31)equipped witha digital camera (OLYMPUS EVOLT330) and images were taken. The isolate was deposited in the Leibniz Institute DSMZ -German CollectionofMicroorganisms and Cell Cultures, Braunschweig, Germany, under the accession number DSM 104095.

Pathogenicity tests
An identi ed isolate of N.dimidiatumKSA based on PCR, anamorph morphology and DNA sequence comparisons was used for thepathogenicity trial to ful l Koch ' s postulates.The 7-day-old incubated isolate grown on aPDA medium at 25°C was used for inoculations.Six-month-old lebbeck tree seedlings were chosen for pathogenicity tests under growth chamber conditionsof 28°C and 15 h daylight. The height of the trees was approximately 90 cm and the diameters of the main stems approximately 10 mm. The seedling trees were allowed toacclimatizeunder thegrowth chamber conditionsfor one month before the inoculations were performed.A sterile cork borer was used to pick up6-mm-diameterdiscs of the PDA medium with the fungal growth ofN.dimidiatumKSA isolates including mycelia. The inoculation was performed on thestems of 10 trees by removing the bark 5 cm above soil levelto disclose the cambium.Five trees were also inoculated with a sterile PDA medium and kept as controls. The areas of inoculation were wrapped tightly with plastic para lm(Pechiney Plastic Packaging) to prevent drying and contamination. Six weeks post inoculation, the bark of the inoculated trees was removed from 5 cm above and below the wounded area and small samples from all inoculated and non-inoculated trees wereplated on a PDA medium for fungal isolation.

Host range studies
The tree species used for the host range studies were selectedbased on ecological importance to the environment of Saudi Arabia andtheir potential susceptibility to the pathogen. Ten healthy seedling treesof Ficus infectoria, Moringa oleifera, Casuarina cunninghan, Enterlobium cyclocorpum, Eucalyptus tereticornis andFicus nitida agedabout six monthswere collected from the forests nurseryofthe College of Agriculture and Veterinary Medicine farm. The seedling trees were grown in plastic pots lled with a 2:1 mixture of sand and clay soil. The seedling trees were maintainedunder growth chamber conditions at 26 o C and 30-60% relative humidity and irrigated once a week. Eight out of the ten trees of each species were inoculated using the identi ed isolate of N.dimidiatumKSA as described previously and the remaining two seedlings of each species were left uninoculated for comparison. Monitoring for the development of symptoms was done weekly. Fungal re-isolation was carried out to verify Koch's postulates.

DNA extraction and PCR sequencing for identi cation of isolates
Pure cultures were maintained on 2% malt extract agar and harvested directly from the plates. Genomic DNA was extracted using the MasterPure™ Yeast (Epicentre, for Fungi and Yeasts) DNA Isolation Kit. The strain was PCR ampli ed and sequenced, targeting the partial gene of the translation elongation factor alpha 1 using primers EF1-728Fand EF1-986R {4}.
The PCR mixtures contained Taq and PCR buffer (Takara Bio Inc., Kusatsu, Japan), 5pM of each primer (Euro ns),200 µM dNTPs (Roche), 40-200 ng of genomic DNA and nuclease-free water. The PCR was performed with an initial denaturation step for 2 min at 94°C, followed by 25-35 cycles of denaturation for 1 min at 94°C, 45-s primer annealing, and elongation for 1 min at 72°C. The quality of the PCR amplicons was checked in 1.2% agarose gel stained with GelStar (Lonza, Switzerland) under UV light using a 1 kb ladder (Gene Ruler 1, Thermo Scienti c, Germany). The amplicons were puri ed using the QuiaQuick puri cation kit (Quiagen, Germany). Sequences were generated with an AB 3730 DNA analyzer (Applied Biosystems, Foster City, CA) and the AmpliTaq FS Big Dye terminator cycle sequencing kit. To type strain references when appropriate,all sequences were used as queries in the GenBank and MycoBank sequence similarity search tool BLAST [http://blast.ncbi.nlm.nih.gov/Blast.cgi] with default stringency and restrictions.

Phylogenetic analysis
Phylogenetic relationships were assessed using the ARB software package (Ludwig et al. 2004). All sequences were aligned using Fast Aligner/ClustalW and Muscle (Edgar 2004) implemented in ARB V1.06. All alignments were thoroughly examined and manually optimized according to primary and secondary structure information calculated by ARB. Ambiguously aligned nucleotide characters were excluded manually prior to phylogenetic analyses. The jModeltest 2.1.1 (Darriba et al. 2012) was used for the selection of the model of nucleotide substitution that best t the sequence data, employing the Akaike Information Criterion (Posada and Buckley 2004). Maximum Likelihood analyses were performed with ARB using RAxML(Randomized Accelerated Maximum Likelihood) 7.0.3 (Stamatakis 2006) applying the GTRGAMMAmodel of sequence evolution for the combined data set. Searches were performed with random sequence additions and 100 replicates. Branch support was tested with 1000 replications on bootstrapped data sets.

Symptomatology and fungal isolation
A survey of the lebbeck trees planted as avenue shade on the main campus of Qassim University in Buraydah in central Saudi Arabia revealed that more than 80% of the trees were wilted and dead. Symptoms of dieback, root rot, stem canker and decline were apparent (Fig. 1a). These symptoms included yellowing branches and browning of leaves and subsequent leaf fall (Fig. 1b). Consequently, rapid death of the branches and cracking of the main trunkswere observed (Fig. 1c).A black mass of fungal spores could be observed on the trunk (Fig. 1f)which soon spread by wind or water to other,healthy trees.The bark of the main roots presentedthe same symptoms.The fungus N.dimidiatumwas isolated from the cracking stems of the ten representative symptomatic lebbeck trees exhibiting dieback. The isolate and its relation to other species of Botryosphaeriaceaewere later con rmed by DNA sequencing.

Culture characteristics and morphology
The colonies on the PDA media were initially whitish, butgradually turned blackish afterthree days (Fig. 2a). They were atand attained radial growth with threadlike forms onthe margin in 3-5 days at 25°C.For inducing the sporulation, an identi ed single spore culture was placed on the surface of 2% water agar medium using a sterile casuarina needle and incubated at 25°C.A chain of cylindrical or spherical arthroconidia appeared on the branched aerial mycelium.Seen under microscopy, the hyaline conidia freed from pycnidia weretruncate at the base,immersed,dark to darkbrown with smooth thick walls, at rst aseptate andlater becoming septatewith central dark brown septa (Fig. 2b). Characteristic of the synasexual morph of coelomycetes, the stromatic conidiomata seen were immersed,lately decrepitate, dark brown or black, spherical, 2-3 mm in diameter with thick, black cell layers and irregular outer walls, while the inner walls were thin and hyaline (Figs. 2c, d).

Pathogenicity tests
Theinoculated lebbeck tree seedlings were assessed after tendays post inoculation. The N.dimidiatumelicited symptoms similar to those shown on the trees naturally infected. Ten days after inoculation, symptoms were starting to appear on four out of eight stems of the inoculated seedlings.Firstly, leaves and branches developed chlorosis, startingwith old leaves, and subsequent leaf fall, and four weeks later, all eight inoculated lebbeck tree seedlings werewilted and bareof leaves,while the uninoculated control seedlings showed no symptoms under test conditions. Re-isolation of the fungus from the stems of the infected lebbeck seedlingsperformed on PDA media to accomplish Koch's postulates revealed the fungus to be N.dimidiatum (Fig. 2).

Host range
The different host plant species examined exhibited symptoms as follows.In Ficus infectoria, all seedling treesinoculated with N. hyalinum showedchlorosis in the leaves followed by necrosis two weeks after inoculation. Canker of the main stem extending to the crown was observed. Thereafter,all the seedlings were completely wilted. At the inoculation site under the bark,a sooty coat of black spores was found coming off in layers (Fig. 3a). In Moringa oleifera, symptoms were seen ten days after inoculation.Chlorosis of the leaves and then leaf fall developed in 50% of the inoculated seedlings. A month later, 80% of the seedlings were completely wilted, while the controls remainedfree of any symptoms (Fig. 3b).In Enterlobium cyclocorpum, leaves of the inoculated seedlings initially became pale and developed chlorosis and necrosis followed byleaf dropthree weeks after inoculation.In ve to six weeks, 75% of the seedlings inoculated under the bark of the stem were completely wilted, whereas the control seedlings did not show any symptoms (Fig. 3c). TheCasuarina equstifolia, Eucalyptus tereticornis and Ficus nitidaseedlings did not produce any symptoms under test conditions and were indistinguishable from the uninoculated seedlings (Fig. 3d).
Re-isolation from the infected seedlings on PDA media con rmed that N. hyalinum was the prevalent fungal pathogen isolated; however, the uninoculated seedlings DNA sequencing and phylogenetic analysis Sequencing of the elongation factor alpha 1 gene region and phylogenetic analysis con rmed that our isolate wasN. dimidiatum (Penz.) Crous & Slippers, a member of the family Botryosphaeriaceae (Fig. 4).Some of the closest BLAST hits for our isolate were identi ed as N. hyalinum. According to Huang et al. {18}, N. hyalinum is synonymous with N. dimidiatum due to the con-speci city of the two.

Discussion
When rst observed, it was striking that most of the lebbeck trees of different ages planted on the main campus of Qassim University at Al-Mulayda (Buraydah), Qassim, central Saudi Arabia, were wilted or dead. Initially, the trees exhibited symptoms ofyellowing and chlorosis on branches and leaves, which turned brown and subsequently suffered leaf fall followed by dieback, stem canker and decline (Fig. 1a). Consequently, quick death of the branches and cracking of the main trunkswere observed. The trunksexhibited a black mass of fungal spores through the cracking bark (Fig. 1c) In the present study, for the rst time, a new isolate of N.dimidiatumKSA wasisolated from infected trees andreported as causing dieback and mortality of lebbeck trees in Saudi Arabia based on symptomatology, phytopathogenicity, host range studies, DNA sequencing, phylogenetic analysis and morphological characteristics of the coelomycetous asexual morph with stromatic dark brown to black, black and erumpent conidiomata, and ellipsoidal to oval and hyaline conidia. The fungus Natrrassiamangifra has a wide geographical range in Africa, Asia, and North and South America {31}. The fungus was rst recorded by Nattrass{24}as causing rapid death on deciduous trees in Egypt, by Sutton  Based on phylogenetic analysis of the elongation factor alpha 1 gene region of the DSM104095 isolatewith 70 othersequencesof the members of Botryosphaeriaceae available in the NCBI/GenBank, it wasdemonstrated thatN. dimidiatumtogether with the other veisolates in the genus, speci callyN. dimidiatum,constitute a separate clade in the tree reconstruction (Fig. 4). All sequences of isolates within this cluster are more closely related to each other than to other members of Botryosphaeriaceaeor other species of the genus Neoscytalidium. Some of the closest BLAST hits for our isolate were identi ed asN. El Gamal et al.{9} reported that thelebbeck trees (A. lebbeck) imported from India to Saudi Arabia as an ornamental tree were well acclimatized to the hot environmental conditions of the central region of Saudi Arabia. It was clear that water stress, high temperatures and wind had in uenced the incidence and severity of the disease {22}. The extreme climate of the central region of Saudi Arabia in addition to stress factors may have contributed to the susceptibility of the lebbeck trees to the dieback disease associated with N. dimidiatum. This is the rst report of N. dimidiatumassociated with lebbeck dieback in Saudi Arabia.
Over the next 50-100 years, climatic change is expected to exert a passive in uenceon the extent of forest land in Saudi Arabia. An increase in the frequency of natural phenomena such as drought, sand storms, re and ood will lead to increased dieback in forests and woodlands, spread of diseases, changesin type and number of species, a drop in productivity and a reduction in biodiversity {6}. Figure 2 NeoscytalidiumdimidiatumKSA(a)Culture of N. hyalinumon PDA after three daysincubation at 25°C.