Diversity of fungal communities in the soil of three nurseries in Tlemcen (North-West of Algeria)
Abstract and Figures
Fungi play a central role in most ecosystems and seem to dominate the microbial biomass in soil habitats, where they are important decomposers and occupy a notable position in the natural carbon, nitrogen and phosphorus cycles. Despite the fact that fungal species have been studied in several contexts, the diversity of some soil fungal species in nurseries is still unknown. This study aimed to explore the diversity of fungal species occurring in nursery soil of Tlemcen province (North-west of Algeria) and to test the antagonist effect of some isolates of Trichoderma sp. on species of the genus Pythium and those of Diplodia. Soil and root samples from young seedlings showing symptoms of oomycete infection and other fungal were collected in three nurseries of Tlemcen region. Following a soil baiting using fresh ornamental leaves, three Phytophthora species were isolated and identified based on morphology and microscopic observation. About the others groups, the dilution method using physiological water, revealed the presence of significant fungal biodiversity on the PDA medium. Twelve fungal species and five oomycetes have been isolated and identified, namely: D. sapinea, Lasio. exigua, F. oxysporum, Aspergillus sp., Penicellium sp., Mucor sp., Trichoderma sp., Alternaria sp. P. cinnamomi, P. gonapodyides, P. ramorum, Py. ultimum and Py. Polare. Significant antagonist activity was recorded for isolates of Trichoderma sp. against Pythium sp. and Diplodia sp.
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Oak decline has been a serious problem in Mediterranean countries since the
beginning of the twentieth century. The invasive pathogen P. cinnamomi has been associated
with intense mortality of oak trees in the Iberian Peninsula, southern France and Italy.
Although oak decline has been reported also in North Africa, P. cinnamomi was never
recovered in any of the surveyed stands, and tree decline has been associated with other
diseases, pests, climate and human intervention. The aim of this work was to investigate the
occurrence of Phytophthora species and their possible involvement in the decline of cork oak
trees in Algeria. A declining cork oak forest located along the Mediterranean northwest coast
was surveyed between 2015-2016. Symptomatic trees showed thinning and crown dieback,
withering of leaves and death. Root rot and extensive loss of both lateral small woody roots
and fine roots were observed. To isolate Phytophthora, roots and soil samples collected from
18 declining and 8 asymptomatic cork oak trees were baited using fresh cork oak leaves.
Isolations were made on SMA, a selective medium for Phytophthora spp. Isolates were
identified using morphological analysis and DNA-based techniques. Phytophtora cinnamomi
was the most frequent species isolated and three other Phytophthora species identified as
P. gonapodyides, P. multivora and P. quercina were detected. Comparative aggressiveness of
these Phytophthora species was assessed on 3-month-old seedlings of Quercus afares,
Q. canariensis, Q. ilex and Q. suber. This is the first study looking at the diversity of
Phytophthora pathogens in Algerian oak forests.
During surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy
and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based
on nuclear ITS, ß-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two
species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species,
P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here
as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth
and very slow growth rates at their respective optimum temperature. They differed from each other and from
related species by a unique combination of morphological characters, cardinal temperatures, and growth rates.
Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated
in soil infestation trials.
Most Phytophthora hybrids characterized to date have emerged from nurseries and managed landscapes, most likely generated as a consequence of biological invasions associated with the movement of living plants and germplasm for ornamental, horticultural and agricultural purposes. Presented here is evidence for natural hybridization among a group of five closely related indigenous clade 6 Phytophthora species isolated from waterways and riparian ecosystems in Western Australia. Molecular characterization of hybrids consisted of cloning and sequencing two nuclear genes (ITS and ASF), sequencing of two further nuclear loci (BT and HSP) and of two mitochondrial loci (COI and NADH). Additionally, phenotypic traits including morphology of sporangia and optima and maxima temperatures for growth were also determined. In most cases the nuclear genes were biparentally and in all cases the mtDNA were uniparentally inherited, indicating hybrid formation through sexual crosses. Some isolates bear the molecular signature of three parents suggesting additional hybrid events, although it cannot be determined from the data if these were sequential or simultaneous. These species and their hybrids co-exist in riparian ecosystems and waterways where their ability for rapid asexual proliferation would enable them to rapidly colonize green plant litter. The apparent ease of hybridization could eventually lead to the merging of species through introgression. However, at this point in time, species integrity has been maintained and a more likely scenario is that the hybrids are not stable evolutionary lineages, but rather transient hybrid clones.
Titration of microorganisms in infectious or environmental samples is a corner stone of quantitative microbiology. A simple method is presented to estimate the microbial counts obtained with the serial dilution technique for microorganisms that can grow on bacteriological media and develop into a colony. The number (concentration) of viable microbial organism is estimated from a single dilution plate (assay) without a need for replicate plates. Our method selects the best agar plate with which to estimate the microbial counts, and takes into account the colony size and plate area that both contribute to the likelihood of miscounting the number of colonies on a plate. The estimate of the optimal count given by our method can be used to narrow the search for the best (optimal) dilution plate and saves time. The required inputs are the plate size, the microbial colony size, and the serial dilution factors. The proposed approach shows relative accuracy within ±0.1log10 from data produced by computer simulations. The method maintains this accuracy even in the presence of dilution errors of up to 10% (for both the aliquot and diluent volumes), microbial counts between 10(4) and 10(12) colony-forming units, dilution ratios from 2 to 100, and plate size to colony size ratios between 6.25 to 200.
Unlabelled:
In this paper we give an account of the genera and species in the Botryosphaeriaceae. We consider morphological characters alone as inadequate to define genera or identify species, given the confusion it has repeatedly introduced in the past, their variation during development, and inevitable overlap as representation grows. Thus it seems likely that all of the older taxa linked to the Botryosphaeriaceae, and for which cultures or DNA sequence data are not available, cannot be linked to the species in this family that are known from culture. Such older taxa will have to be disregarded for future use unless they are epitypified. We therefore focus this paper on the 17 genera that can now be recognised phylogenetically, which concentrates on the species that are presently known from culture. Included is a historical overview of the family, the morphological features that define the genera and species and detailed descriptions of the 17 genera and 110 species. Keys to the genera and species are also provided. Phylogenetic relationships of the genera are given in a multi-locus tree based on combined SSU, ITS, LSU, EF1-α and β-tubulin sequences. The morphological descriptions are supplemented by phylogenetic trees (ITS alone or ITS + EF1-α) for the species in each genus.
Taxonomic novelties:
New species - Neofusicoccum batangarum Begoude, Jol. Roux & Slippers. New combinations - Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, Botryosphaeria ramosa (Pavlic, T.I. Burgess, M.J. Wingf.) A.J.L. Phillips & A. Alves, Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips, Cophinforma mamane (D.E. Gardner) A.J.L. Phillips & A. Alves, Dothiorella pretoriensis (Jami, Gryzenh., Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Dothiorella thailandica (D.Q. Dai., J.K. Liu & K.D. Hyde) Abdollahz., A.J.L. Phillips & A. Alves, Dothiorella uruguayensis (C.A. Pérez, Blanchette, Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Lasiodiplodia lignicola (Ariyawansa, J.K. Liu & K.D. Hyde) A.J.L. Phillips, A. Alves & Abdollahz., Neoscytalidium hyalinum (C.K. Campb. & J.L. Mulder) A.J.L. Phillips, Groenewald & Crous, Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. & Pennycook) A.J.L. Phillips & A. Alves, Sphaeropsis eucalypticola (Doilom, J.K. Liu, & K.D. Hyde) A.J.L. Phillips, Sphaeropsis porosa (Van Niekerk & Crous) A.J.L. Phillips & A. Alves. Epitypification (basionym) - Sphaeria sapinea Fries. Neotypifications (basionyms) - Botryodiplodia theobromae Pat., Physalospora agaves Henn, Sphaeria atrovirens var. visci Alb. & Schwein.
An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re-isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.
Between 2008 and 2011, severe dieback associated with root and collar rot was reported on Arbutus unedo in several sites in Sardinia, Italy. Isolations from infected tissues and rhizosphere soil samples consistently yielded a Phytophthora species. It was initially identified as Phytophthora cinnamomi var. parvispora Krober and Marwitz by comparing morphological features with the original description and the internal transcribed spacer (ITS) sequences with those present in GenBank. A multigene phylogeny based on DNA sequence data from two nuclear (ITS and b-tubulin) and two mitochondrial (cox1 and cox2) gene regions combined with extensive morphological and physiological properties of these isolates, including the ex-type culture of P. cinnamomi var. parvispora, demonstrates that this taxon is unique and it is redesignated here as Phytophthora parvispora sp. nov. Although morphologically similar to P. cinnamomi, P. parvispora differs by its smaller-
sized sporangia, chlamydospores, oogonia and oospores, higher oospore wall index, single-celled antheridia, higher minimum and maximum temperatures for growth and faster growth at optimum temperature. In the phylogeny, P. parvispora falls within Phytophthora ITS clade 7a, grouped in a well-supported clade sister to P. cinnamomi. In pathogenicity tests, P. parvispora and P. cinnamomi were equally aggressive towards A. unedo seedlings. The possible geographic origin of P. parvispora is also discussed.
The interaction between Trichoderma harzianum and sclerotia of the soilborne plant pathogen Sclerotium rolfsii was studied by scanning and transmission electron microscopy (SEM and TEM, respectively) to assess the potential role of enzymatic hydrolysis in the antagonistic process. SEM investigations revealed that hyphae of T. harzianum grew abundantly on the sclerotial surface and formed a dense branched mycelium that appeared to establish contact with the outer host cells through a thin mucilage. Observation of cross-sections of parasitized sclerotia by light microscopy showed that hyphae of the antagonist multiplied on the sclerotial surface and displayed the ability to penetrate the rind. Growth of the antagonist in the rind layer was mainly intracellular, and host-wall penetration was achieved by means of constricted hyphae. Ultrastructural observations showed that Trichoderma growth and development coincided with extensive host cell alterations, such as retraction and aggregation of the cytoplasm and vacuole breakdown. In the invaded outer rind cells, host cell walls apparently were not altered, as judged by their preserved structure. In contrast, cell breakdown due to host cell-wall disruption was observed more frequently in inner rind cells adjacent to medullary cells. Ingress of T. harzianum hyphae in the medulla was characterized mainly by a change in the mode of growth from intra- to intercellular. Trichoderma hyphae did not penetrate the medullary cells, although the latter showed pronounced alterations, such as cytoplasm disorganization and aggregation. The use of wheat germ agglutinin/ovomucoid-gold complex for localization of chitin monomers resulted in regular labeling of both host and antagonist cell walls, even when sclerotia were massively colonized. Chitinolytic degradation at a distance from the site of Trichoderma penetration was never observed. There was no indication of cell-wall disorganization in either the host or the antagonist, as shown by the regular distribution of labeling, even in zones of penetration by constricted Trichoderma hyphae. In the medulla, gold labeling was regularly distributed over the thick host cell walls, even when the medullary cells showed obvious signs of disorganization. When ultrathin sections of parasitized sclerotia were incubated with the gold-complexed β-1,3-glucanase for localization of β-1,3-glucans, a regular distribution of gold particles was observed over the walls of both outer and inner rind cells, even when these exhibited a disorganized cytoplasm that was reduced to a few remnants of aggregated material. Incubation with gold-complexed lipoprotein lipase yielded a pattern of labeling similar to that obtained with gold-complexed β-1,3-glucanase. Gold particles were evenly distributed over the host cell walls in the rind layer.