Melons

The CGMMV experimental host N. benthamiana and cucurbits hosts (watermelon, melon, cucumber and bottle gourd) were used for VIGS of the PDS gene by CGMMV vectors in this study. Watermelon (Zhengkang 2), melon (Baimei), cucumber (Jinyan 4) and bottle gourd (Yongzhen1) seeds were obtained from Zhengzhou Fruit Research Institute (Zhengzhou, China), Xinjiang Academy of Agricultural Sciences (Xinjiang, China), Tianjin Academy of Agricultural Sciences (Tianjin, China) and Ningbo Academy of Agricultural Sciences (Ningbo, China), respectively. All cucurbit seeds were soaked in sterile water for 3–4 h at 50 °C, then placed in Petri plates containing wetted filter cotton gauze at 28 °C in darkness until seeds were germinated. Germinated seeds were planted into pots with nutrient matrix and grown in a growth chamber under 16 h light at 28 °C/8 h dark at approximately 22 °C. The same conditions were used to grow inoculated plants (see below) with CGMMV vectors.

The study was conducted in a section (450 km²) of the municipality of Pampa del Indio (25°55′S 56°58′W), Province of Chaco, Argentina (Figure 1A, inset), located in the humid (east) Chaco, close to the transition to the dry (west) Chaco. Based on data collected by the Chagas disease control program of Chaco, which indicated high infestation levels, we selected the municipality of Pampa del Indio as this project's location. Based on an exploratory survey throughout the rural area of Pampa del Indio, in which we inspected for triatomine infestation a systematic sample (11%) of the district's houses, we selected a well-defined section with slightly higher infestation than the rest and more than 300 adjacent houses isolated ≥1 km from the nearest villages outside of the selected area.
The study area included 353 houses and several public buildings of 13 neighboring rural villages (Figure 1). The climate is continental, warm, with rain mainly in summer. Annual mean temperature is 22.8°C (mean minimum and maximum, 16.9 and 29.3°C). Annual rainfall historically has been 954 mm, although in 2008 and 2009 a severe drought affected the region. The landscape is flat and comprises mainly a mosaic of patches of crops mixed with native dry forest that has undergone various degrees of degradation, and with occassional water bodies and marshes.
The two main ethnic groups are Creole and Toba. Tobas represent 24% of the 1,187 inhabitants of the study area and occupy 16% of the houses. Creoles are of European descent and usually had a high degree of mixing with indigenous people generations ago. Most Creoles migrated to the area during the last 50–100 years from nearby provinces or from Europe. Tobas –the only indigenous group in the area– were traditionally nomadic or semi-nomadic hunter-gatherers. Following local colonization in the 1920s they began to rely increasingly on agriculture, temporary informal jobs, and state-run welfare programs a few decades ago [25] . Local authorities reported having approximately 5,000 beneficiaries of welfare programs among the ∼14,000 inhabitants in the whole municipality, both Toba and Creoles. Rural residents live mostly on a subsistence economy, and may grow cotton, corn, pumpkins and water-melons or raise livestock (mainly goats, but also cows and occassionally sheep). The nearest hospital is in Pampa del Indio town (∼5,000 inhabitants), 10–45 km away from the study area by dirt roads. The last community-wide insecticide spraying campaign conducted by vector control personnel was carried out in 1996, except for a few houses treated by villagers or hospital staff in 2006, and no specific or systematic educational campaigns regarding Chagas disease or its transmission were performed in the area.
The study area encompassed 327 inhabited house compounds, including all of its domestic and peridomestic sites. There were 26 uninhabited or abandoned houses and 37 public buildings (including 11 schools, five primary health care centers, and several temples and community centers). A house compound encompassed the domicile and all sites within the peridomestic area (i.e., peridomicile) –usually a latrine, a storeroom, a kitchen, an oven, one or more corrals, and one or more sites for chickens and other poultry (trees, coops, nests) (Figure 2A,B). A site was any individual structure built and/or given a defined use by householders which might provide refuge for bugs. Ecotope (a categorical variable) was defined as a site characterized by some typical structure and use (e.g., domicile, storeroom, chicken coop, etc.). Nests were frequently found within a distinctive structure called ‘nidero’ (from the Spanish for nest, ‘nido’, Figure 2C), consisting of an elevated shelf made of wood or sometimes bricks where chickens and, occasionally, turkeys or ducks nested. Most domiciles were mud-and-thatch huts with corrugated metal or tarred-cardboard roof.

The hyphal development was visualized using the Trypan Blue staining technique (Koch and Slusarenko, 1990 (link)). In brief, the PM-infected melon leaves were soaked in Trypan Blue staining solution and immediately heated in 100 °C water for 2–5 min. After allowing the solution to cool down to room temperature, the staining solution was discarded and the leaves were decolored using a 2.5 mg/ml chloral hydrate solution, which was replaced every 24 hours until the leaves were completely decolored.

The amino acid sequences of the MLO gene family in melon were also analyzed by using the online analysis software Multiple Expectation Maximizations for Motif Elicitation (MEME) (http://meme-suite.org/), and ten motifs were identified for subsequent analysis. Meanwhile, a gene structure map was obtained by using the online analysis software of an upgraded gene feature visualization server, “Gene Structure Display Server (GSDS), v2.0” (http://gsds.gao-lab.org/) (Hu et al., 2015 (link)).