Conidial Color Research Articles

Hidden diversity of Pestalotiopsis and Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) species allied with the stromata of entomopathogenic fungi in Taiwan

Pestalotiopsissensu lato, commonly referred to as pestalotiopsis-like fungi, exhibit a broad distribution and are frequently found as endophytes, saprobes and pathogens across various plant hosts. The taxa within pestalotiopsis-like fungi are classified into three genera viz. Pestalotiopsis, Pseudopestalotiopsis and Neopestalotiopsis, based on the conidial colour of their median cells and multi-locus molecular phylogenies. In the course of a biodiversity investigation focusing on pestalotiopsis-like fungi, a total of 12 fungal strains were identified. These strains were found to be associated with stromata of Beauveria, Ophiocordyceps and Tolypocladium in various regions of Taiwan from 2018 to 2021. These strains were evaluated morphologically and multi-locus phylogenetic analyses of the ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α) and tub2 (beta-tubulin) gene regions were conducted for genotyping. The results revealed seven well-classified taxa and one tentative clade in Pestalotiopsis and Neopestalotiopsis. One novel species, Pestalotiopsis manyueyuanani and four new records, N. camelliae-oleiferae, N. haikouensis, P. chamaeropis and P. hispanica, were reported for the first time in Taiwan. In addition, P. formosana and an unclassified strain of Neopestalotiopsis were identified, based on similarities of phylogeny and morphology. However, the data obtained in the present study suggest that the currently recommended loci for species delimitation of pestalotiopsis-like fungi do not deliver reliable or adequate resolution of tree topologies. The in-vitro mycelial growth rates of selected strains from these taxa had an optimum temperature of 25 °C, but growth ceased at 5 °C and 35 °C, while all the strains grew faster under alkaline than acidic or neutral pH conditions. This study provides the first assessment of pestalotiopsis-like fungi, associated with entomopathogenic taxa.

Uncovering the Morphological and Genetical Heterogeneity of Pyricularia oryzae (cooke) sacc. in Southwestern Region of Bangladesh

Significant yield losses are reported due to blast disease caused by Pyricularia oryzae across all rice growing areas of the world. Though, management strategies reduce disease significantly but blast epidemics are still common. This research was aimed to determine the variability among the 19 Pyricularia oryzae isolates of South-Western regions of Bangladesh. Morphological variability was determined based on nine morphological components like front mycelial color, reverse mycelial color, growth behavior, conidial length (μm), conidial width (μm), conidial color, radial growth at 12 days (mm), 14 days (mm) and 16 days (mm) after incubation. Cluster analysis considering nine morphological components showed five distinct groups. Front and reverse mycelial color, growth behavior and conidial length played more than 80% role in generating variation among the nine tested components. Higher genetic variation was also detected among the 19 isolates of P. oryzae. Here, larger numbers of amplified DNA fragments was formed by Primers UCB-155 which showed distinct polymorphism. Between two clusters, Cluster II consists of 18 isolates that were originated from different origin but having maximum 94% genetical similarity. Cluster I contain only one isolates PO-16. From pair wise distance matrix, maximum distance (80%) was found between PO-16 and PO-03 isolates and minimum distance (6%) was obtained between the isolates PO-11 and PO-12. Results of this experiment revealed that, higher genetic diversity among isolates of P. oryzae existed in the south western region of Bangladesh but there has no direct relation with genetic diversity and geographical origin.

Re-Evaluating Botryosphaeriales: Ancestral State Reconstructions of Selected Characters and Evolution of Nutritional Modes.

Botryosphaeriales (Dothideomycetes, Ascomycota) occur in a wide range of habitats as endophytes, saprobes, and pathogens. The order Botryosphaeriales has not been subjected to evaluation since 2019 by Phillips and co-authors using phylogenetic and evolutionary analyses. Subsequently, many studies introduced novel taxa into the order and revised several families separately. In addition, no ancestral character studies have been conducted for this order. Therefore, in this study, we re-evaluated the character evolution and taxonomic placements of Botryosphaeriales species based on ancestral character evolution, divergence time estimation, and phylogenetic relationships, including all the novel taxa that have been introduced so far. Maximum likelihood, maximum parsimony, and Bayesian inference analyses were conducted on a combined LSU and ITS sequence alignment. Ancestral state reconstruction was carried out for conidial colour, septation, and nutritional mode. Divergence times estimates revealed that Botryosphaeriales originated around 109 Mya in the early epoch of the Cretaceous period. All six families in Botryosphaeriales evolved in the late epoch of the Cretaceous period (66-100 Mya), during which Angiosperms also appeared, rapidly diversified and became dominant on land. Families of Botryosphaeriales diversified during the Paleogene and Neogene periods in the Cenozoic era. The order comprises the families Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae and Saccharataceae. Furthermore, current study assessed two hypotheses; the first one being "All Botryosphaeriales species originated as endophytes and then switched into saprobes when their hosts died or into pathogens when their hosts were under stress"; the second hypothesis states that "There is a link between the conidial colour and nutritional mode in botryosphaerialean taxa". Ancestral state reconstruction and nutritional mode analyses revealed a pathogenic/saprobic nutritional mode as the ancestral character. However, we could not provide strong evidence for the first hypothesis mainly due to the significantly low number of studies reporting the endophytic botryosphaerialean taxa. Results also showed that hyaline and aseptate conidia were ancestral characters in Botryosphaeriales and supported the relationship between conidial pigmentation and the pathogenicity of Botryosphaeriales species.

The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of ΔCfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development (CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation (CfCBP1 and CfCHS7), pectin degradation (CfPelA and CfPelB), sexual development (CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis (CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

Genome sequences of 24 Aspergillus niger sensu stricto strains to study strain diversity, heterokaryon compatibility, and sexual reproduction.

Mating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24 strains were sequenced and analyzed revealing an average of 6.1 ± 2.0 variants/kb between Aspergillus niger sensu stricto strains. The genome sequences were used together with available genome data to generate a phylogenetic tree revealing 3 distinct clades within Aspergillus niger sensu stricto. The phylogenetic tree revealed that both MAT1-1 and MAT1-2 mating types were present in each of the 3 clades. The phylogenetic differences were used to select for strains to analyze heterokaryon compatibility. Conidial color markers (fwnA and brnA) and auxotrophic markers (pyrG and nicB) were introduced via CRISPR/Cas9-based genome editing in a selection of strains. Twenty-three parasexual crosses using 11 different strains were performed. Only a single parasexual cross between genetically highly similar strains resulted in a successful formation of heterokaryotic mycelium and subsequent diploid formation, indicating widespread heterokaryon incompatibility as well as multiple active heterokaryon incompatibility systems between Aspergillus niger sensu stricto strains. The 2 vegetatively compatible strains were of 2 different mating types and a stable diploid was isolated from this heterokaryon. Sclerotium formation was induced on agar media containing Triton X-100; however, the sclerotia remained sterile and no ascospores were observed. Nevertheless, this is the first report of a diploid Aspergillus niger sensu stricto strain with 2 different mating types, which offers the unique possibility to screen for conditions that might lead to ascospore formation in A. niger.

Major involvement of two laccase genes in conidial pigment biosynthesis in Aspergillus oryzae.

Wild-type strains of Aspergillus oryzae develop yellow, yellow-green, green, or brown conidia. Previous reports suggested that the conidiation initiates with the biosynthesis of a yellow pigment YWA1 from acetyl-CoA by a polyketide synthase encoded by wA (AO090102000545). This is followed by the conversion to other pigment by a laccase encoded by yA (AO090011000755). Based on orthologous pathways in other Aspergilli, it is reasonable to hypothesize that in addition to yA, AO090102000546 encoding laccase and AO090005000332 encoding Ayg1-like hydrolase play a role in A. oryzae conidial pigment biosynthesis. However, the involvement of these two genes in conidial pigmentation remains unclear. In this study, we tested this hypothesis by assessing the conidial colors of both disruption and overexpression mutants to verify whether AO090102000546 and AO090005000332 were associated with the conidial pigmentation. Observation of single, double, and triple disruptants of these three genes suggested that conidial pigments were synthesized by two laccase genes, AO090011000755 and AO090102000546, whereas Ayg1-like hydrolase gene AO090005000332 was proven to have no obvious association with the synthesis. This was corroborated by observing the phenotype of each overexpression mutant. Interestingly, AO090005000332 overexpression mutant produced smoky yellow-green conidia, different from the wild-type strain. Thus, the AO090005000332-encoded protein is likely to maintain the enzymatic activity. However, the expression level was observed to be one-third of that of AO090102000546 and one-seventh of that of AO090011000755. Consequently, apparent lack of obvious contribution of AO090005000332 to conidial pigmentation could be attributed to its low expression level. Expression analysis indicated similar profiles in several wild-type strains. KEY POINTS: • Conidial pigment biosynthesis after YWA1 mainly involves two laccases in A. oryzae. • Ayg1-like hydrolase in A. oryzae is not obviously involved in conidial pigmentation. • Conidial color is deemed dependent on expression level of two laccases and hydrolase.

Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers.

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.

Pathogenicity, Host Range and Influence of Temperature, Humidity and pH Levels on the Growth of Fusarium oxysporum f.sp. lentis

Background: Lentil is a vital nutritional source of protein in several parts of the world including India. The crop is susceptible to wilt which is a devastating soil-borne disease induced by the fungus Fusarium oxysporum f. sp. lentis. Insight of the potential threat Fusarium wilt can pose to lentils, a present study done on pathogenicity, host range and influence of temperature, humidity and pH levels on the growth of F. oxysporum f. sp. lentis.Methods: Ten isolates FOL-01 to FOL-10 of F. oxysporum f. sp. lentis (Fol) were isolated from wilted lentil plants that collected from different major lentil growing parts of Rajasthan. During 2017-18 a pathogenicity test was tested in pot house condition by seed and soil inoculation techniques for all isolates and epidomological factors evaluated in vitro conditions.Result: Results indicated that the Fol isolates represent a single race but differ in their aggressiveness on the susceptible cultivar L9-12. Pathogenicity test revealed clearly that Fol was associated with wilt symptoms and were pathogenic to lentil plants. A maximum percent disease incidence of 70.00 was showed by isolate FOL-02 in soil inoculation technique. In the morphological and cultural characterization, all the ten isolates showed various character in conidial frequency, colony color and growth pattern. Twenty plant species were tested to know the host range of Fol, out of these lentil, chickpea and pea show positive reaction with the pathogen. The influence of temperature, relative humidity and pH on the growth and sporulation of Fol was studied under in vitro conditions. Maximum mycelial growth and sporulation of the Fol were observed at 30°C, 6.0 pH and 60% relative humidity.

Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment.

Conidia are asexual spores and play a crucial role in fungal dissemination. Conidial pigmentation is important for tolerance against UV radiation and contributes to survival of fungi. The molecular basis of conidial pigmentation has been studied in several fungal species. In spite of sharing the initial common step of polyketide formation, other steps for pigment biosynthesis appear to be species-dependent. In this study, we isolated an Aspergillus flavus spontaneous mutant that produced yellow conidia. The underlying genetic defect, a three-nucleotide in-frame deletion in the gene, AFLA_051390, that encodes a copper-transporting ATPase, was identified by a comparative genomics approach. This genetic association was confirmed by disruption of the wild-type gene. When yellow mutants were grown on medium supplemented with copper ions or chloride ions, green conidial color was partially and nearly completely restored, respectively. Further disruption of AFLA_045660, an orthologue of Aspergillus nidulans yA (yellow pigment) that encodes a multicopper oxidase, in wild type and a derived strainproducing dark green conidia showed that it yielded mutants that produced gold conidia. The results placed formation of the gold pigment after that of the yellow pigment and before that of the dark green pigment. Using reported inhibitors of DHN-melanin (tricyclazole and phthalide) and DOPA-melanin (tropolone and kojic acid) pathways on a set of conidial color mutants, we investigated the involvement of melanin biosynthesis in A. flavus conidial pigment formation. Results imply that both pathways have no bearing on conidial pigment biosynthesis of A. flavus.

Aspergillus hancockii sp. nov., a biosynthetically talented fungus endemic to southeastern Australian soils.

Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.

Two novel Aspergillus species from hypersaline soils of The National Park of Lake Urmia, Iran

Two novel Aspergillus species, one belonging to the section Terrei and the other to section Flavipedes, were isolated from hypersaline soils of The National Park of Lake Urmia (Iran) and are here described as Aspergillus iranicus and Aspergillus urmiensis. A polyphasic taxonomic approach comprising extrolite profiles, phenotypic characters and molecular data (beta-tubulin, calmodulin and ribosomal polymerase II second largest subunit gene sequences) was applied to determine their novel taxonomic status. Aspergillus iranicus (CBS 139561T) is phylogenetically related to A. carneus, A. niveus, A. allahabadii and A. neoindicus, and it can be differentiated from those species by a unique extrolite pattern (citrinin, gregatins, and a terrequinone) and its conidial colour. Aspergillus urmiensis (CBS 139558T) shares a most recent common ancestor with A. templicola. The former species produces globose vesicles, and those of A. templicola are predominantly elongate. The Aspergillus urmiensis isolates produce several uncharacterized extrolites. Two other strains obtained during this study reside in a clade, together with the type strain of A. movilensis (CCF 4410T), and are identified accordingly. Based on the phylogenetic data presented in this study, A. frequens is reduced to synonymy with A. micronesiensis and A. mangaliensis is considered to be a synonym of A. templicola.

Revising the Schizoparmaceae: Coniella and its synonyms Pilidiella and Schizoparme.

The asexual genera Coniella (1918) and Pilidiella (1927), including their sexual morphs in Schizoparme (1923), have a cosmopolitan distribution and are associated with foliar, fruit, leaf, stem and root diseases on a wide variety of hosts. Species of these genera sometimes occur as secondary invaders of plant tissues infected by other organisms or that are injured by other causes. Several studies published over the last few decades had conflicting ideas as to whether Coniella, Pilidiella and Schizoparme should be regarded as synonymous or as separate genera. The present study aims to resolve the generic classification of these genera through phylogenetic analyses of the concatenated alignment of partial LSU nrDNA, rpb2, ITS nrDNA and tef1 sequence data of 117 isolates, combined with their morphology. Results revealed that all strains cluster in a single well-supported clade. Conidial colour, traditionally the distinguishing character between Coniella and Pilidiella, evolved multiple times throughout the clade, and is not a good character at generic level in Schizoparmaceae. The three genera should therefore be regarded as synonymous, with the older name Coniella having priority. Furthermore, this study delineated 13 new species, and new combinations were proposed for a further 15 species.

Morphological Characterization of Biocontrol Isolates of <i>Trichoderma</i> to Study the Correlation between Morphological Characters and Biocontrol Efficacy

The morphological characterization was carried out for 5 isolates of Trichoderma harzianum and 7 isolates of Trichoderma viride and tested for their biocontrol efficacy. The isolates belonging to T.harzianum were analogous in colony colour, culture smell, mycelial colour, conidiation, conidial shape, conidial wall and conidial colour. Correspondingly the isolates of T.viride showed certain similarity in colony colour, colony edge, culture smell, conidiophore branching, conidial wall, conidial colour and chlamydospores. Inter specific differences through cluster analysis based on morphological characters grouped the twelve isolates into three major clusters where all the isolates of T.harzianum formed a single cluster while the isolates of T.viride were bifurcated into two groups. The clustering was substantiated by similarity index which showed maximum similarity among T.harzianum isolates with only less than 20% variation among themselves. Similarly the clusters having isolates of T.viride also had less variation within them. The biocontrol efficacy of these twelve isolates of Trichoderma was experimented by dual culture test under laboratory condition and there existed some relation between the biocontrol efficacy of these isolates and morphology.

Morphological Characterization of Biocontrol Isolates of <i>Trichoderma</i> to Study the Correlation between Morphological Characters and Biocontrol Efficacy

The morphological characterization was carried out for 5 isolates of Trichoderma harzianum and 7 isolates of Trichoderma viride and tested for their biocontrol efficacy. The isolates belonging to T.harzianum were analogous in colony colour, culture smell, mycelial colour, conidiation, conidial shape, conidial wall and conidial colour. Correspondingly the isolates of T.viride showed certain similarity in colony colour, colony edge, culture smell, conidiophore branching, conidial wall, conidial colour and chlamydospores. Inter specific differences through cluster analysis based on morphological characters grouped the twelve isolates into three major clusters where all the isolates of T.harzianum formed a single cluster while the isolates of T.viride were bifurcated into two groups. The clustering was substantiated by similarity index which showed maximum similarity among T.harzianum isolates with only less than 20% variation among themselves. Similarly the clusters having isolates of T.viride also had less variation within them. The biocontrol efficacy of these twelve isolates of Trichoderma was experimented by dual culture test under laboratory condition and there existed some relation between the biocontrol efficacy of these isolates and morphology.

Diversity of Mycotoxigenic Fusarium armeniacum Isolated from Rice Grains at Harvest Time in Korea

A total of 509 rice panicle samples were collected at harvest time from fields in 8 provinces from 2010 to 2014. One hundred five grains per sample were plated on potato dextrose agar and 6,658 Fusarium isolates were obtained; among them, 67 were identified as Fusarium armeniacum by sequencing the translation elongation factor () and confirmed by their morphological and cultural characteristics. Considerable variation in conidial size, colony color and sequences was observed among the fungal isolates. The ability of 24 F. armeniacum isolates to produce T-2 and HT-2 toxin in potato sucrose agar was determined using liquid chromatography-mass spectrometry. Twenty one isolates produced T-2 and HT-2 toxin, resulting in varying toxin levels among the isolates. The results show that Korean isolates of F. armeniacum have diversity with respect to morphological, cultural, genetic, and toxigenic properties.

First Report of Alternaria longipes Causing Leaf Spot of Potato Cultivar Sante in Pakistan.

Potato (Solanum tuberosum) is one of the most important vegetable crops worldwide, including Pakistan. During surveys from November to February of 2011 to 2013 in Sahiwal (Punjab), a severe leaf spot disease, new to farmers, was recorded. Symptoms consisted of 1- to 3-mm diameter black circular necrotic spots and appeared on the leaves of 2- to 3-week-old plants. Disease incidence was ~70 to 80%. This disease was localized to few fields in Sahiwal on potato variety Sante and to our knowledge, this has not been found on other areas or potato varieties in Pakistan. Fungi were isolated from randomly selected diseased plants. Ten infected plants were brought to the laboratory in sterilized polyethylene bags. One infected leaf per plant was selected for pathogen isolation. Infected parts of leaves were cut into ~2 mm2 pieces. Leaf pieces were surface sterilized for 1 min with 0.5% sodium hypochlorite and then inoculated aseptically onto 2% malt extract agar (MEA) (Sigma, Dorset, UK) and incubated at 25 ± 2°C for 3 to 4 days in the dark. Hyphal tip transfer from emerging colonies was performed to obtain pure cultures. Initial microscopic examination of pure fungal colonies revealed Alternaria as the likely causal organism. For morphology-based identification, five isolates from separate infected leaves were grown on MEA as well as potato carrot agar (PCA) for 7 days. All isolates showed similar morphological characters including dusty greenish black, floccose colonies with regular and smooth margins reaching 3 to 4.5 cm in diameter on MEA and sporulation with well-defined zones of growth. Aerial hyphae produced long branches that bore lateral chains of 1 to 7 conidia. Conidia were pointed at the tip, ovoid or ellipsoid, ranged from 18 to 40 × 5 to 12 μm with 4 to 8 transverse and 0 to 1 longitudinal septa. No conidial beak was present. Conidial color darkened from dull olive to brown as the culture matured. Based on morphology, the pathogen was identified as Alternaria longipes (1). A pure culture of a fungal pathogen was submitted to First Fungal Culture Bank of Pakistan (FCBP1355) for future reference. To confirm the morphology-based identification, the rDNA internal transcribed spacer (ITS) nucleotide sequence was amplified using ITS1 forward and ITS4 reverse primers (2). The amplicon of 537 bp was sequenced and submitted to GenBank under accession KJ806191. A BLASTn search using the KJ806191 sequence revealed it to be 99% identical to around 20 different strains of A. longipes deposited in GenBank including leaf spot pathogens of another Solanaceaeous member, Nicotiana tabacum (AY154684) and Asteraceous plant, Atractylodes macrocepha (JQ004404). Pathogenicity testing was performed in the greenhouse at 30 ± 2°C. Pots (16 × 9 cm) were filled with sterilized soil. Since spores of Alternaria sp. are known to survive in soil or plant debris, soil was sterilized and inoculated with 106 spore suspension of the isolated pathogen before sowing the potato seeds. Control pots were not inoculated. Approximately 10 days after plant germination, the previously observed disease symptoms appeared on leaves and A. longipes was re-isolated from the necrotic areas of leaves, thus fulfilling Koch's postulates. Plants in control treatments were asymptomatic. Pathogenicity tests were repeated three times. To our knowledge, this is the first report of A. longipes leaf spot of potato cultivar Sante from Pakistan. However, the distribution of this disease is confined to the area where it was observed, but it could be a threat for potato crop if not managed timely.

First Report of Leaf Spot of Rice Caused by Alternaria arborescens in Pakistan.

Rice (Oryza sativa) is one of the most profitable and popular cereal crops in Pakistan. In July 2012, symptoms consisting of circular, black, necrotic spots, 2 to 4 mm in diameter, were observed on leaves of a commonly grown rice cultivar, Basmati-198, in private rice fields at Lahore (Punjab). This disease was observed later on rice cultivar KSK-133 grown at Faisalabad (Punjab) during the same cropping season. Disease incidence was ~35% and 25% for Basmati-198 and KSK-133, respectively. To our knowledge, the pathogen was confined only in these areas and cultivars and was not present on other rice varieties or crops. Ten infected plants were selected randomly from each field of two rice cultivars and one infected leaf for each of the 10 infected plants was selected for the isolation of fungal pathogen. Necrotic lesions were cut into pieces of ~2 mm2, surface-disinfected with 0.5% sodium hypochlorite, placed on 2% malt extract agar (MEA) (Sigma, Dorset, UK), and incubated at 25 ± 2°C for 4 to 5 days. Emerging fungal colonies were transferred aseptically to fresh MEA petri plates for purification. Alternaria spp. were consistently recovered from infected leaves. Three isolates per variety were selected for detailed morphological studies. Each isolate was grown at 25°C on MEA and potato carrot agar (PCA) for 7 days. All isolates displayed similar morphological features including black radiate, floccose colonies with irregular margins, 6 to 7 cm in diameter on MEA and 2 to 3 cm with 1 to 2 pairs of concentric growth rings on PCA. Conidial chains were not crowded with 1 to 10 conidia per branch and bearing several lateral branches. Conidiophores were tapering and narrow, 40 to 200 × 2 μm. Conidia were ovoid within a size range of 10 to 30 × 5 to 14 μm, with 1 to 5 transverse and 1 longitudinal septum. Conidial color darkens from a dull tan to a medium brown as the culture matures. Based on morphology, the pathogen was identified as Alternaria arborescens (1). A pure culture of the pathogen was deposited in First Fungal Culture Bank of Pakistan (FCBP) with accession FCBP1351. Identification based on morphology was verified by sequencing the internal transcribed spacer (ITS) region. For this, a DNA fragment of ~650 bp was amplified using total genomic DNA as template and ITS1 and ITS4 primers (2). The nucleotide sequence of the ITS region was submitted to GenBank under accession KF679683. Comparison of the sequence with those in GenBank revealed that the sequence was 99% identical with A. arborescens, isolate ALT-242 (KC415808), causing Eucalyptus leaf spot in India and strain STE-U4345 (AF404667), a causal pathogen of apple core rot in South Africa. Pathogenicity testing was performed on both cultivars. One-month-old plants grown in greenhouse were sprayed with 10 ml of spore suspension (2 × 105 spores/ml) as well as 10 ml of this spore suspension in soil at the time of sowing. Control plants were sprayed with sterilized water. The plants were covered with plastic bags for 48 h and kept under observation for 2 weeks in a glasshouse at 30 ± 2°C. Lesions appeared on leaves after 10 days of inoculation whereas control plants remained healthy. Pathogenicity tests were repeated in triplicate. Similar disease symptoms and re-isolation of A. arborescens fulfilled Koch's postulates. To our knowledge, this is the first report of A. arborescens leaf spot of rice in Pakistan. At present, the distribution of this disease is limited to the fields where it was observed.

Comparative Study of Biological Agents, Trichoderma harzianum (Th- Azad) and Trichoderma viride (01PP) for Controlling Wilt Disease in Pigeon Pea

The paper aims to study the morphological, physiological, molecular characterization and bio-formulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315, an effective fungicide and a biological control agent too that protects the plants and seeds from other pathogenic fungi. The physiologic study is done in an attempt to find the effective management of the disease caused by soil borne pathogens. Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 isolated from the infected soil samples of Pigeon pea fields and grown at preferable temperatures, pH and different solid and liquid culture media. Most preferable temperature for the growth and sporulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 has been observed up to 30oC (210.5 mg dry weight of mycelium). A detailed morphology of the strain is done in this study including colony growth rate, colony color, colony edge, mycelial form, conidiation, conidiophore branching, conidial wall, conidial color, etc. The molecular characterization of the strain is carried out using an 18S rRNA gene sequence with the help of a universal Internal Transcribed Spacer marker that gives an amplicon of a total of 1173 base pairs and 546 bp of the 18S rRNA gene was sequenced and used for the identification of isolated fungal strains that is later sequenced and allotted with Gene Bank Accession no. JX119211 and KC800922 respectively. To check the presence of endochitinase gene in two of the potential strains of Trichoderma species viz. T. harzianum (Th Azad) and T. viride 01PP an ech42 primer was used. A talc based bio-formulation has been prepared with this strain where the population of the spores was found to decline after 180 days.

LAC2 Encoding a Secreted Laccase Is Involved in Appressorial Melanization and Conidial Pigmentation in Colletotrichum orbiculare

Both Colletotrichum and Magnaporthe spp. develop appressoria pigmented with melanin, which is essential for fungal pathogenicity. 1,8-Dihydroxynaphthalene (1,8-DHN) is believed to be polymerized to yield melanin around the appresorial cell wall through the oxidative activity of laccases. However, no 1,8-DHN laccase has yet been identified in either Colletotrichum or Magnaporthe spp. Here, we report a laccase gene, LAC2, that is involved in the appressorial melanization of Colletotrichum orbiculare, which causes cucumber anthracnose. LAC2 encodes a protein with a signal peptide and has high homology to fungal laccases. The conidial color of lac2 mutants is distinct from that of the C. orbiculare wild type, and the mutants are nonpathogenic. Notably, the mutant appressoria are defective in melanization, and a host invasion assay showed that the appressoria are nonfunctional. LAC2 was induced during appressorial melanization. These results suggest that LAC2 oxidizes 1,8-DHN in the appressoria. The LAC2 homologues of other fungi located in the same phylogenetic clade as LAC2 fully complemented the lac2 mutants. Interestingly, a LAC2 homologue, located in a different clade, complemented the conidial pigmentation but not appressorial melanization of the mutants, suggesting that the LAC2 function in appressorial melanization might only be conserved in laccases of the LAC2 clade.

Appearance of a new leaf rot disease on common ice plant

An unknown disease abruptly appeared on hydroponic cultures of common ice plant (Mesembryanthemum crystallinum) in the greenhouse, causing catastrophic damage. Although the symptoms of the plant were unlike typical Botrytis lesions on leaves and stems of other plants, fungi isolated from the necrotic lesions on the plant were similar to genus Botrytis in terms of conidial shape, colony color and nature. A representative isolate, Ice-2, caused similar symptoms on the host plants after inoculation with conidia, and the same fungus was isolated from the lesions. The conidia and conidiophores of Ice-2 were morphologically similar to those of B. cinerea but not to those of B.allii, B. fabae or B. squamosa. The tested Botrytis fungi grew at temperatures between 5 and 30°C. Ice-2 grew faster than the others at the lower end of the temperature range. Ice-2 was also more virulent than B. cinerea (Bay-1) in artificial inoculations, especially on common ice plant leaves. The glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) sequence of Ice-2 was determined and compared with those from four Botrytis species. The gene sequence of Ice-2 appeared to be identical to that of B. cinerea. In leaf tests on common ice plant and kidney bean, the diseases caused by Ice-2 and Bay-1 were controlled equally well by the primary Botrytis fungicides. Based on the results of the present studies, Ice-2 is thought to be Botrytis cinerea Person: Fries.