The ability of Brevibacillus brevis to influence development of disease on tomato caused by Fusarium oxysporum f.sp. lycopersici was investigated using plants raised in Petri dish microcosms and in pots in the glasshouse. Development of symptoms on both microcosm-and glasshouse-raised tomato plants was markedly reduced in co-inoculations of F. oxysporum f.sp. lycopersici with B. brevis, compared with inoculations with the pathogen alone. Moreover, co-inoculations resulted in significant growth boosting effects on the plants, with increases in plant height in microcosms and in total root lengths in glasshouse-raised plants. In microcosm-raised plants, the carrier used to inoculate seed with B. brevis, either carboxymethyl cellulose (CMC) or vermiculite, had no effect on the persistence of the biological control agent on roots in the absence of inoculation with the pathogen. By contrast, numbers of B. brevis recovered from the rhizosphere and rhizoplane of inoculated plants in microcosms were four orders of magnitude lower than in plants treated with B. brevis alone. Moreover, higher numbers of B. brevis CFU were re-isolated from the rhizosphere of plants arising from CMC-coated seed, than vermiculite-coated seed. The carrier had no effect on disease control. Inhibition of conidial germination and germtube extension of F. oxysporum f.sp. lycopersici by cellfree filtrates of B. brevis cultures varied significantly depending on the culture medium used for suspension. These results indicate that B. brevis is a potential biological control agent for reducing the impact of F. oxysporum f.sp. lycopersici on tomato.
BACKGROUND Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression (Woo et al., 2015). Comprehensive data from one of our STING agonists was presented at the AACR Tumor Immunology meeting in 2017. Here we describe CRD5500, a versatile next generation compound that can be dosed either by intra-tumoral route, systemically (IV or SC) or as an antibody drug conjugated to Trastuzumab (ADC). The ADC approach allows for tumor targeting through systemic delivery with low systemic exposure of the payload and should result in a higher Therapeutic Index (TI) in the clinic. METHODS We have described the discovery of small molecule activators of human STING that generate potent anti-tumor innate and adaptive responses in recently published patents. CRD 5500 was evaluated in (1) in reporter gene assays on HEK293T cells containing stably transfected hSTING polymorphs, (2) by immunoblots to confirm pSTING, pTBK1, pIRF3, (3) by real-time PCR and ELISA monitoring induction of Type I cytokines in human tumor cell lines, isolated human PBMCs and dendritic cells (hMo-DC). Direct STING binding was demonstrated by (1) Cellular Thermal Shift Assays (CETSA) and (2) in vitro kinase assay. Anti-tumor activity of CRD5500 was demonstrated by multiple routes in syngenic tumors containing human STING. Libraries of linkers were used to permit conjugation of CRD5500 with Trastuzumab. Prior to conjugation, penultimate constructs (Pre-Ab) were screened for their ability to preferentially release CRD5500 in tumor homogenate with reduced plasma exposure. Antibody drug conjugates were synthesized at Abzena, UK and were tested in HER2 expressing cells to assess release of free payload . RESULTS Treatment of an isolated cell free system containing purified recombinant STING and TBK1 leads to STING phosphorylation confirming that CRD-5500 is a direct STING binder. Evidence of direct binding also comes from CETSA. CRD-5500 activates all five common hSTING variants with agonist EC50 comparable to CDNs in the pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 leads to phosphorylation of IRF3, TBK1 and STING. CRD-5500 causes the maturation of hDCs and the release of innate and adaptive inflammatory cytokines such as IFNβ and TNFα from hDCs and hPBMCs. In vivo administration of CRD5500 IT or systemically caused tumor regression in CT26 syngeneic tumors containing human STING. CRD5500 demonstrated the abscopal effect and when combined with check point blockade dramatically reduced tumor mass. Interestingly, when Pre-Ab constructs that were not conjugated to antibody were delivered IV, they combined with an anti-CTLA4 antibody to eliminate tumors in the hSTING.CT26 syngenic tumor model. Conjugation with Trastuzumab was demonstrated and the ADC thus formed was screened in HER2 expressing cell lines. CONCLUSIONS The ability of hSTING agonists to generate Type 1 IFN anti-tumor immune responses makes them a promising therapeutic option either as a single agent or in combination with other therapies. CRD5500 is a small molecule STING agonist that can be delivered in multiple formats making it well suited for further development as an anti-cancer agent. Citation Format: Monali Banerjee, Sourav Basu, Sandip Middya, Ritesh Shrivastava, Rajib Ghosh, David C. Pryde, Dharmendra Yadav, Gopal Bhattacharya, Thanilsana Soram, Kavita Puniya, Ritika Raina, Vijay Kadam, Sujay Garai, Priti Sharma, Anuj Singh, Vaibhav Shinde, Nidhi Rawat, Anindita Middya, Arghyotri Sinha, Sunita Chandel, Ganesh Narisipuram, Abhisek Chatterjee, Nagaswamy Mane, Arjun Surya. CRD5500: A versatile small molecule STING agonist amenable to bioconjugation as an ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-061.
Abstract:A new foliar disease of toona tree has been reported during continuous surveys conducted at Solan district of Himachal Pradesh during August, 2015 to December, 2015. On the basis of morphological characteristics of the fungus, the fungus was identified as Rhytisma acerinum, which produces symptoms on upper surface of the leaves as numerous small, superficial and blackish lesion which later gives the similar appearance as given by tar. Tar spot of the toona tree produces the ascomata whose size ranges from 14-16 µm with average of 15µm, asci length varied from 60-80 x 9.5-10.2 µm and size of ascospores varied from 52-58 x 1.8-2.0 µm. Tar Spot disease of leaves of Toona in Himachal Pradesh is the first report of its occurrence and more work is needed so that the disease could remain in manageable level.
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