First Report of Anthracnose on Alfalfa Caused by Colletotrichum linicola in Serbia.

Alfalfa (Medicago sativa L.) is economically the most important forage crop in Serbia. In July 2009, alfalfa plants showed symptoms characteristic of anthracnose disease ("shepherd's crook") including wilting and death of the upper portion of the stems. Anthracnose of alfalfa has been reported to be caused by Colletotrichum trifolii or C. destructivum (2). Alfalfa plants with anthracnose symptoms were collected in Srpska Crnja, South Banat District, Serbia. Infected tissue samples were surface disinfected with 5% sodium hypochlorite for 2 min and washed three times for 5 min in sterile distilled water. Surface sterilized tissue was transferred to sterile filter paper and placed on potato dextrose agar (PDA), and incubated at 24°C in the dark for 10 days (1). Developing colonies were light to dark olive green. In cultures on PDA medium, acervuli were formed. Conidia from acervuli were released in mucous masses that were orange to cream-pink in color. Conidia were hyaline, aseptate, straight with one end pointed and the other slightly rounded, measuring 12.5 to 25.0 × 2.5 to 7.5 µm (mean 19.83 × 4.42 µm). After 5 days, numerous setae were formed. The setae were slightly darker at the bottom and lighter at the top, septate with 3 septa. Setae dimensions were 100 to 185.5 × 2.5 to 5 µm (average 160.9 × 3.12 µm). The isolated fungus was designated Coll-44. Stems of 30 7-week-old plants were spray-inoculated in the laboratory with an aqueous suspension of conidia (106 spores per ml; 10 ml per plant) harvested from 7-day-old cultures grown on PDA. The plants and two non-inoculated check plants were placed in a greenhouse and a covered with plastic bags at 25°C in darkness. After 48 h, plastic bags were removed from the all plants. All plants were watered once a day. Symptoms were observed 10 days after inoculation. No symptoms were observed on non-inoculated plants. In the greenhouse, all 30 inoculated plants became diseased with anthracnose symptoms after 10 days. Coll-44 was consistently re-isolated from diseased stem tissue. Koch's postulates were fulfilled by re-isolation from inoculated alfalfa plants. Pure culture of the Coll-44 isolate was deposited in the public collection of CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (specimen no. CBS 3263). Partial sequences of the internal transcribed spacer regions-ITS (GenBank Accession No. JX908364) and betatubulin-TUB2 gene (KJ556347) were amplified and sequenced from extracted fungal DNA with primer pairs ITS1-ITS4 (4) and T1-Bt2b (3), respectively. ITS sequence of the Coll-44 isolate showed 100% nucleotide identity to the GenBank accessions JQ005765 and AB046609 of C. linicola. TUB2 sequence of isolate Coll-44 showed 99.6% nucleotide identity with the GenBank accession JQ005849 of C. linicola isolate CBS 172.51. To our knowledge, this is the first report of C. linicola causing alfalfa anthracnose in Serbia. References: (1) A. P. Baxter et al. S. Afr. J. Bot. 2:259, 1983. (2) K. D. Hyde et al. Fungal Divers. 39:1, 2009. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Identification and Characterization of Eutypa lata on Grapevine in Serbia.

In the period from late May 2004 to late May 2010, grapevine (Vitis vinifera L.) between 11 and 22 years old was observed for the incidence of symptoms of shortened shoots and zigzag internodes, with tiny, chlorotic leaves, torn and bended edges, with necrosis along the edges of leaves and dead internervous tissue. These symptoms are conspicuous especially when the vine is in the F phases of development. Later, in the course of vegetation, the dying of infected shoots and branches or covering of symptoms by a new foliage mass is perceived. Foliar symptoms are initially confined to one arm of infected vines; however, as the disease progresses, symptoms may spread throughout the entire vine. If a cross-section is made of an infected trunk, the canker appears as a wedge-shaped area of discolored wood spreading to the center of the trunk. In the period from 2004 to 2010, disease incidence was high, approaching 20%, and decline-affected cordons, vine branches, or whole plants was very high, resulting in losses of more than 35%. The loss created by a decline in grapevine quality is not included in this estimate. In this study, the causal agent was diagnosed as Eutypa lata (anamorph Libertella blepharis), on the basis of morphology of conidia of a Libertella anamorph on a 4- to 6-week-old culture on PDA (1) and by molecular identification. Molecular identification was performed by PCR and RFLP analysis and supplemented by sequence analysis. Total DNA was isolated from cultured mycelia of fungi using CTAB extraction protocol. PCR reaction was performed by universal ITS1/ITS4; the primer pair and RFLP patterns were determined after restriction with AluI (3). For specific identification of E. lata, the primer pair Lata 1/Lata 2.2 (2) were used and the 385-bp fragment was detected from analyzed isolates. Five selected isolates were purified and a fragment encompassing ITS1, ITS2, and 5.8S rDNA gene was sequenced. Sequences were deposited in the NCBI database under Accession Nos. JQ041699, JQ041700, JQ041701, JQ041702, and JQ041703. Sequence comparisons revealed high nucleotide identity among isolates (99.6 to 100%). When aligned with other E. lata isolates retrieved from the NCBI database, Serbian isolates show the highest nucleotide identity with the isolates from North America (AY462541, AY462540, AY662393, AY662392) and Australia (EU835166, EU835163, EU835162, EU835161, EU835160, EU835159, EU835156). A pathogenicity test was performed in February 2006 in a greenhouse at room temperature (approximately 22°C) and included inoculation rooted cuttings of grapevine (cultivars Cabernet Sauvignon, Prokupac, and Drenak) plants by mycelium. Agar plugs containing mycelium were inserted into 5 mm diameter holes drilled in the main stem of the rootlings and sealed by wrapping with Parafilm. Uninoculated control vines treated with a sterile agar plug were included in the experiment. Foliar symptoms and discoloring of wood beneath and above the inoculation site, inoculated plants, was observed. Reisolation and reinoculation were performed 27 months later, and 54 months later the pathogenicity test was confirmed (4). To our knowledge, this is the first report of death of infected cordons of grapevine by E. lata in Serbia. References: (1) D. A. Glawe et al. Mycotaxon 2:123, 1982. (2) P. Lecomte et al. Appl. Environ. Microbiol. 66:4475, 2000. (3) P. E. Rolshausen et al. Plant Dis. 88:925, 2004. (4) M. Sosnowski et al. Aust. N.Z. Grapegrower Winemaker 493:14, 2005.

A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing.

Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched in signaling molecules. The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. However, little is known about the regulation of the initial raft redistribution and whether negative regulatory signaling pathways target this phase of cellular activation. We used natural killer (NK) cells as a model to investigate the regulation of raft redistribution, as both positive and negative signals have been implicated in the development of their cellular function. Here we show that after NK cells form conjugates with sensitive tumor cells, rafts become polarized to the site of target recognition. This redistribution of lipid rafts requires the activation of both Src and Syk family protein tyrosine kinases. In contrast, engagement of major histocompatibility complex (MHC)-recognizing killer cell inhibitory receptors (KIRs) on NK cells by resistant, MHC-bearing tumor targets blocks raft redistribution. This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase. These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

Risk stratification in myeloma by detection of circulating plasma cells prior to autologous stem cell transplantation in the novel agent era.

The impact of circulating plasma cells (CPCs) prior to autologous stem cell transplantation (ASCT) for multiple myeloma has not been defined in the novel agent era. We evaluated the impact of pre-transplant CPCs, detected by six-color flow cytometry in patients undergoing early ASCT on post-transplant response, progression-free survival (PFS) and overall survival (OS). CPCs were detected in 162 out of 840 (19.3%) patients, with the median number of CPCs being 58 per 150 000 events. Ninety-nine percent of patients had received proteasome inhibitor and/or immunomodulator-based induction. The incidence of post-transplant stringent complete response (sCR) in the subgroups with and without CPCs was 15% and 38%, respectively, (P<0.001). The median PFS in the subgroups with and without CPCs was 15.1 (95% confidence interval (CI), 12.5-17.8) and 29.6 months (95% CI, 26.2-32.8), respectively, and the median OS was 41.0 months (95% CI, 32.6-58.2) and not reached (NR) (95% CI, 99.1-NR), respectively, (P<0.001 for both). On multivariate analysis for OS, factors independently predictive of mortality were the presence of CPCs (hazard ratio (HR) 2.5; 95% CI, 1.8-3.6; P<0.001) and sCR post transplant (HR 0.4; 95% CI, 0.2-0.6; P<0.001). Presence of CPCs prior to transplant has a high prognostic impact and should be prospectively validated in clinical trials.

Analysis of recombinant Plum pox virus (PPV) isolates from Serbia confirms genetic homogeneity and supports a regional origin for the PPV-Rec subgroup.

The recent observation of the frequent occurrence of natural recombinant Plum pox virus (PPV) isolates has led to the identification of a distinct PPV subgroup, named PPV-Rec. The diversity, origin and geographical spread of the recombinant PPV isolates belonging to this subgroup remain, however, relatively poorly known. In an effort to further our understanding of these isolates, eight PPV isolates from Serbia, the country from which the first such recombinant (PPV-o6) originated, were characterized. Depending on the genomic region targeted by different typing assays, seven of the eight isolates tested presented discrepancies in their typing behavior. Sequence analysis of the (Cter)NIb-(Nter)CP region confirmed the recombinant nature of these seven isolates which all presented an identical recombination breakpoint identical to previously characterized PPV-Rec isolates. Biological indexing and immunoblot analysis provided indications that asymptomatic infection of the GF305 peach indicator and migration of the coat protein as a double-band in immunoblots may represent conserved and discriminating properties of PPV-Rec isolates. The genetic diversity of PPV-Rec isolates from former Yugoslavia (Serbia, Bosnia and Herzegovina) was estimated to be twice as large as that of the PPV-Rec isolates obtained from all other countries to date (Albania, Bulgaria, Czech republic, Germany, Hungary and Slovakia). These last results are consistent with the hypothesis that former Yugoslavia is the center of dispersion of PPV-Rec. Taken together, the results presented here provide further evidence for the wide distribution and temporal genetic stability of these natural PPV recombinant isolates and provide for the first time a possible scenario for their dispersion throughout central and eastern Europe.

Regulation of NK cell-mediated cytotoxicity by the adaptor protein 3BP2.

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.

Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy.

We describe here a novel strategy to enhance the in vivo efficacy of replicating adenovirus therapy, using coinjection of plasmid DNA encoding a fusogenic viral glycoprotein. The combination of fusogenic membrane glycoprotein (FMG)-induced tumor cell fusion and infection with replicating adenovirus effectively treats even large established tumors at doses of plasmid DNA and virus that alone are ineffective. Adenoviral infection appears to increase the transduction of the tumor cells to a modest degree thereby boosting the FMG-mediated component of the therapy. Simultaneously, syncytial formation enhances the therapeutic effects of viral infection by increasing spread of adenoviral particles through the tumor cell population and by increasing titer of virus released from the tumor cells. This effect is due probably to release of intracellular viral particles upon tumor cell death and also to increased levels of E1A protein within syncytia, whose increased metabolic rate is associated with enhanced levels of protein expression. Cotransduction of tumor cells with replicating adenovirus and FMG-expressing vectors could either be combined within single replicating vectors or could be used in strategies using separate administration of two components, both at lower doses than required for either therapy alone.

SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors.

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.

Cutting edge: a role for the adaptor protein LAT in human NK cell-mediated cytotoxicity.

Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcgammaRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-gamma. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and "natural cytotoxicity," thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.