DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.

Assessment of Injuries Caused by Anastrepha fraterculus (Wied.) (Diptera: Tephritidae) on the Incidence of Bunch Rot Diseases in Table Grape.

Resumo: Anastrepha fraterculus (. Wied) é a principal praga de uvas de mesa (Vitis vinifera) na Região Sul do Brasil. Neste estudo, o objetivo foi investigar o efeito da punção de frutas por fêmeas adultas e infestação larvária por A. fraterculus na ocorrência da doença podridões na uva (cultivar "Itália"). Abstract: Anastrepha fraterculus (Wied.) is the main insect pest of table grapes (Vitis vinifera) in the Southern Region of Brazil. In this study, we aimed to investigate the effect of fruit puncturing by adult females and larval infestation by A. fraterculus on the occurrence of bunch rot disease in the grape (cultivar ?Itália?) by evaluating grapes (a) punctured for oviposition by females of A. fraterculus, sterilized in laboratory with novaluron (40 mg L−1) and further spray-inoculated separately with Botrytis cinerea (1 × 106 conidia mL−1), Glomerella cingulata (1 × 106 conidia mL−1), and bacteria and yeast that cause sour rot (1 × 105 cells mL−1), (b) grapes punctured for oviposition by non-sterilized females with pathogen spraying, (c) grapes with mechanical wounds and pathogen spraying, (d) grapes with no wounds and with pathogen spraying, (e) grapes punctured for oviposition by A. fraterculus chemically sterilized in laboratory with novaluron, (f) grapes punctured for oviposition by A. fraterculus non-sterilized in laboratory with novaluron, (g) grapes with mechanical wounds, and (h) grapes with no sterilization or pathogen spraying. Our data indicated that the mechanical and oviposition wounds caused by A. fraterculus increased the percentage of grapes infected by B. cinerea, G. cingulata, and microorganisms of acid rot. The grape puncturing by A. fraterculus and the mechanical wound allows the penetration of B. cinerea and microorganisms leading to acid rot. We conclude that the fruit fly A. fraterculus may facilitate phytopathogens penetration leading to bunch rots in the table grape Itália.