Materials And Methods

Area of study. Work was carried out in a roselle plantation located in Pozolapa, in the municipal area of Ayutla de los Libres, Guerrero, Mexico (16° 53’ 35.2’’ N, 99° 05’ 59.7’’ O), at an altitude of 275 masl, with a background of a high incidence of aqueous leaf spotting, calyx slight and RCS during two production cycles in the spring-summer of 2017 and 2018. The municipal area presents a sub-humid warm weather (Aw1) (^{García, 2004}). The plantation under study was of one hectare. Planting was carried out on July 4 and 10, 2017 and 2018 respectively, using a handspike, with four to six seeds per shrub, of the genotype known as “Coneja”, at a distance of 1.0 m between furrows and plants. When the plants reached a height of 10 cm, a thinning process was carried out in order to leave two to three plants per shrub, with an approximate density of 25,000 plants ha^-1. Fertilization was carried out using the formula 45-30-20 in two applications (^{Alejo, 2016}). No agrochemicals were applied in the plantation for the control of pests and diseases.

Identification of fungi. In 2017, two leaves and two calyces with sunken necrotic spots in the intermediate section of 14 plants were gathered. These plants were cut into 56 fragments of 2.0 mm each, disinfected with sodium hypochlorite at 1.0% for 30 s and rinsed in three successive changes of sterile distilled water and planted in a potato-dextrose-agar (PDA) culture medium; finally, they were incubated for six days at 26 °C with 12-hour photoperiod. From these colonies in growth, 28 subcultures were obtained and monoconidial cultures were carried out using the technique of scratch in agar at 2%. After six days, the fungal colonies of each sample were identified up to the genus level, using the key by ^{Barnett and Hunter (1998}).

From the isolations carried out, two Phoma colonies were chosen and purified using hyphae tip transfers. For the extraction of DNA, transfers were made to a liquid potato-dextrose medium, incubated for 24 hours while shaking and the mycelium was collected. The DNA extraction was carried out using the method by ^{Dellaporta et al. (1983}); its concentration, quality and purity was quantified in a nanodrop Thermo Scientific^® One, placing 2 uL of DNA per sample. To confirm the identification of the strains, regions of the internal tyranscribed space (ITS) was amplified, using the primers ITS1 / ITS4 (^{White et al., 1990}). PCRs were carried out using the Top Taq Master mix QIAGEN kit, in the Bio-RadT-100^® thermocycler. The conditions for amplification consisted of an initial stage of pre-incubation for two minutes at 95 °C followed by 35 denaturalization cycles for 30 s at 95 °C, hibridation for 30 s at 55 °C and a minute-long extension at 72 °C, and a final extension stage for 10 min at 72 °C (White et al., 1990). The amplified fragments were observed using agarose gel in TAE at 1.0%, stained with GelRed. The fragment of amplified DNA was sent for sequencing in both directions using the method of dideoxynucleotides marked in the 3130 Genetic Analyzer sequencer (Applied Biosystems^®) to the National Agricultural, Medical and Environmental Biotechnology Laboratory of the Instituto Potosino de Investigación Científica y Tecnológica, A.C. (IPICYT). The sequences obtained were compared for their identification with a search of sequences in the database of the gene bank of the National Center for Biotechnology Information (NCBI) using the BLAST algorithm (^{Altschul et al., 1997}; ^{Zhang et al., 2000}).

In order to confirm the pathogenicity of two selected strains, healthy yet susceptible roselle plants of genotypes Ayutla and Tecoanapa were used. The C. cassiicola strain from H. sabdariffa (MF000878.1) was obtained from the mycological collection of the Colegio de Postgraduados Mexico (Dr. Javier Hernández-Morales) and the second strain, identified by morphology as Phoma, was chosen for its identification with ITS markers. Each strain was considered a treatment and the experimental unit was a plant. Six plants of every genotype were inoculated with C. cassiicola, another six per genotype, with Phoma, and three plants were used per genotype as a control (sprayed with sterile distilled water) each one aged 28 days, in a biospace located in the Iguala Experimental Field in 2017 (28 °C and 66% of relative humidity). Inoculation was carried out by spraying the healthy leaves with a spore suspension (6x10⁷ spores/mL for C. cassiicola and 1x10⁷ spores/mL for Phoma. Afterwards, the plants were covered for three days using a plastic bag, disinfected with alcohol. In order to maintain a temperature of 28.9 °C and a humidity of 88%, the first 12 leaves were evaluated to determine the incidence of the symptoms. A second inoculation was carried out when the plants were 77 days old; the temperature was 27.3 °C and a relative humidity of 70%, the flower buds were sprayed with a spore suspension (6x10⁷ spores/mL for C. cassiicola and 10x10⁷ spores/mL for Phoma) and the control treatment was sprayed with sterile distilled water. The first three calyces of each plant were evaluated. The incidence of the disease was calculated by dividing the number of leaves and calyces with symptoms by the total number of leaves and calyces, multiplied by 100. Evaluations were performed on leaves and calyces 3, 5 and 8 days after inoculation. One leaf and calyx from each plant per treatment were planted in PDA using the method described above, and re-isolated it from the tissues with spots, the cultures and their reproductive structures, which were compared with the characteristics of the cultures inoculated originally.

Sampling of spores. From July to November, 2017 and 2018, spores present in the air were captured and counted in the experimental plantation; a Burckard volumetric trap was used, which works with a continuous air suction process, with a record of seven days (^{Gadoury and MacHardy, 1983}). The spore trap was placed in the center of the plantation, 1.7 m above the roselle canopy. The spores drawn in hit a cylindrical drum, covered by Janel sticky tape covered in Vaseline. The tape was cut into 39.5 mm sections, one for each 24-hour period, and placed on a slide. The spore count was in three transects at 400x magnification, calculating the average observed during 24 h. At least five spores were observed, another three transects were counted and the average was calculated to report the concentration of spores accumulated in seven days. Spores were monitored for 133 and 122 days in 2017 and 2018 respectively, and in the phenological stages of growth, development of flower buds, fruition and before harvest.

Weather measurements. The weather factors of rainfall, relative humidity, global radiation and wind speed were recorded in a Davis Vantage PRO2 weather station from July to December in 2017 and 2018 respectively. Using the records of global radiation, the number of sunlight hours per day were recorded.

Monitoring the disease severity. The level of damage by the disease was determined in 25 plants chosen at random. In each one, a leaf and a calyx were chosen per branch on the lower, intermediate and lower sections of the plant, per cardinal point, counting 12 leaves and calyces per plant and with a diagrammatic scale by ^{Ortega-Acosta et al. (2016}), with a range of 0 to 5; in leaves 0=healthy (0% damage) and 5= severe damage (>57% damage); in calyces 0=healthy (0% damage) and 5= severe damage (>77% damage) to determine the severity. The evaluation period for leaves was in September before the emergence of flowers, and for flowers and calyces, in November, before harvesting in 2017 and 2018.

Statistical analysis. The spore capture records were grouped in seven-day periods and analyzed with descriptive statistics and the Spearman correlation (^{SAS, 2010}) between spores accumulated every week and weather data. The Spearman correlation was also used to relate the number of spores accumulated every week with the severity of the disease in leaves and calyces.

Results

Identification of fungi. A white mycelial growth later turned gray, eight days later formed hyalin-colored pycnidial conidiomaths, which then turned dark brown and round, covering the Petri dish. The conidiophores were very dense, thin, simple and sometimes branched. The immature hyalin spores were unicellular, elliptical, they later turned light brown, smooth, ellipsoidal, and measured 10-16.2x 5-7 µm, characteristics which place them in the genus Phoma (^{Barnett and Hunter (1998}). Two representatives were placed in the Collection of Phytopathogenic Fungi of the Phytopathology Lab, Campo Experimental Centro de Chiapas, CIRPAS-INIFAP, as Gro-1701 and Gro-1702. The DNA sequencing of this strain had a similarity of 99% with Coniella diplodiella (=Phoma.diplodiella) (KC771899.1).

The second growth of septated mycelia short, cyllindrical conidiophora and conidia, either individual, terminal or short-chained conidia, with a brown acropetal formation, multicellular, with a thick, colorless exospore and a dark, prominent scar, characteristics coincide with the genus Corynespora (^{Barnett and Hunter, 1998}). The third mycelial growth was fast and abundant, in 1 to 3 days, colored gray to olive greenish, with pycnidial conidiomes that appeared after 13 days, black in color, obpiriform, ostiolated, with ellipsoidal to sub-ovoidal, immature hyaline conidia after 17 days; after 22 days, we observed mature, dark brown, ellipsoidal to ovoidal conidia with longitudinal and irregular striations, morphological characteristics that coincide with the genus Lasiodiplodia (Sutton, 1980; Barnett and Hunter, 1998).

The fungi Coniella sp. y Corynespora sp. Displayed the highest frequency of diseased tissue in leaves isolations with 71.4 and 21.4% respectively, and in calyces, both fungi, with 57.1 and 35.7% respectively (Table 1). The fungus with the least isolation was Lasiodiplodia sp., which was isolated with a low frequency in leaves and calyces, with 7.1%.

The fungus C. diplodiella caused the typical RCS symptoms in the Ayutla and Tecoanapa genotypes, with 88.9 and 22.2% of symptoms in leaves and calyces of the plants, respectively. The fungus C. cassiicola also caused similar RCS symptoms in both genotypes, with 56.3 and 25% of symptoms in leaves and calyces. With both fungi, the symptoms on leaves began with irregular spots and the formation of a red halo and a necrotic center measuring 1-3 mm at 72 and 192 h after inoculation with C. diplodiella and C. cassiicola respectively. The calyces displayed slightly sunken black spots, irregularly shaped, with a diameter of approximately 1-2 mm on days 53 and 91 after the second inoculation with C. diplodiella and C. cassiicola respectively. However, the C. diplodiella isolation was more aggressive on leaves, since it caused a greater incidence on the Tecoanapa and Ayutla genotype, whereas C. cassiicola had a lower incidence and there were even three plants of the Ayutla genotype that displayed no symptoms. By contrast, the incidence of the disease in the calyx was slightly higher, since C. cassiicola had an incidence of 25.0% and C. diplodiella, it was 22.2%. The infection of leaves and calyces was confirmed with these treatments when re-isolating the two fungi from the typical spots.

Spore sampling. From the sample of spores in the air, five genera were identified using their morphological characteristics: Coniella, Corynespora, Lasiodiplodia, Curvularia and Alternaria (^{Barnett and Hunter 1998}). The genera Curvularia, Alternaria and Lasiodiplodia have pathogenic abilities, recognized in several hosts. Lasiodiplodia was reported in fresh roselle calyces by ^{Martínez (2010}), while Ruiz (2014) reported Alternaria in dried calyces. However, in the ethiology of the spotting of roselle calyces, the presence and pathogenicity of Pilidella (=Coniella) diplodiella (^{Correa et al., 2011}), Coniella javanica (Barrón et al., 2019) and Corynespora cassiicola (^{Ortega-Acosta et al., 2015}) have been consistent. This is the reason why this study was focused on these pathogens.

Table 2 shows the abundance of spores from planting to harvest. The genus Coniella displayed 19.3 and 32.3 spores/week in 2017 and 2018 respectively (with a maximum of 223.6 spores/week from September 19 to 25, 2018), with a detection rate of 44.4 and 52.0% for 2017 and 2018 respectively, and an average of 48.2% in both cycles. The genus Corynespora displayed lower concentrations, with 3.9 and 5.3 spores/week in 2017 and 2018 (with a maximum of 12.4 spores/week from November 24 to 30, 2018), with a detection rate of 9.0 and 8.5% in 2017 and 2018 respectively, and an average of 8.7% in both cycles. The Lasiodiplodia spores were less abundant and with a low detection rate in both cycles.

Weather measurements. The highest rainfall levels were 141.2 mm on September 14, 2017 and 71.9 mm on October 10, 2018 (Table 3). The highest temperatures occurred in 2018, with an average of 32.7 °C, whereas in 2017 it was 31.9 °C. The lowest temperature occurred in 2018 with 14.9 °C, and in 2017, it was 15.8 °C. The highest global radiation, of 600.5 W/m², took place on August 11, 2018 and 553.7 W/m² on July 31, 2017. The highest wind speed range reached up to 3.2 km hr^-1 in 2017, and in 2018, it was 2.7 km hr^-1. Mean relative humidity was 86.4 and 84.4% in 2017 and 2018, respectively.

Seasonal distribution of inoculant in the air. The capture of spores in the study area in the period between July and November, 2017 and 2018, displayed the presence of Coniella and Corynespora during the entire planting cycle (Figure 1). The detection of spores began with the emergence of the first true leaves in roselle plants and it continued until the harvest, with the observation of a clear monthly variation.

Figure 1. Weekly distribution of Coniella and Corynespora spores in the air, from July to November, 2017 and 2018. The phenology of roselle is shown below. The typical planting, flowering and harvesting periods in Guerrero are indicated with black lines. 

In July, 2017, 10 days after planting, we registered the presence of Coniella and Corynespora spores, with an average of 20 and 4.5 spores/week, respectively. During vegetative development, the population levels of both fungi were reduced. Coniella, with an average of 0.7 spores/week between August 25 and October 13, 2017, and of 0.2 spores/week between August 29 and September 11, 2018. Corynespora presented slightly higher populations, with an average of 1.9 spores/week between August 25 and October 13, 2017, and of 0.7 spores/week between August 29 and September 11, 2018. However, during the development of flower buds, the fruition and physiological maturity of calyces increased their population levels. Coniella, with an average of 41.2 spores/week between October 13 to November 24, 2017, and of 49.5 spores/week between September 12 and November 30, 2018. Meanwhile, Corynespora registered lower population averages, with 7.1 spores/week between October 13 and November 24, 2017, and 7.2 spores/week between September 12 and November 30, 2018.

Correlation between spores and weather. The concentrations of captures of fungal spores were correlated with the weather variables using Spearman’s test, and the values are shown in Table 4. The individual spore populations from the five genera studied, along with the total of spores, displayed sensitivity to weather variations. Rainfall led to a significant negative effect only on the concentration of the Corynespora inoculant. Temperature was the variable that favored the dynamics of fungi population in the air the most. The highest temperature showed a significant positive correlation with the fluctuations in population of Coniella and Curvularia inoculants and the total of spores, whereas Alternaria displayed a negative correlation, and the minimum temperature, only with the dynamics of the Corynespora population. The average daily temperature displayed a positive correlation with the fluctuation of Coniella and Curvularia spores and the total of spores. Likewise, the maximum wind speed had a negative correlation with the amount of Corynespora spores. In this study, global radiation was another variable that had an influence on the dynamics of the population of fungi in the air; average global radiation presented a positive influence on the dynamics of the Coniella and Curvularia populations and the total spores, while the number of hours of solar radiation displayed a negative influence with the Corynespora spores.

Correlation between the severity of RCS and Coniella and Corynespora spores. In September 2017, the severity of the RCS in leaves reached an index of 2.6 and the concentration of Coniella and Corynespora spores was 121.2 and 9.1 spores/week, respectively, in the las week of said month (Figure 2a). The highest accumulated rainfall level was 936.6 mm and occurred in September. The disease severity in the calyces reached an index of 3.9 in November, and the concentrations of Coniella and Corynespora was 34.7 and 8.3 spores/week, respectively, in the second week of November (Figure 2a), whereas between the second week of October and the third week of November, accumulated rainfall was 9.7 mm. In 2018, the severity of RCS in leaves was high, with 3.2 on the scale in September, and the concentration of Coniella and Corynespora spores was higher, with 223.6 and 9.1 spores/week, respectively, in the last week of said month (Figure 2b); in September, there was an accumulated rainfall of 48.3 mm. The severity of the disease in the calyces reached an index of 3.55 in November, whereas the concentrations of Coniella and Corynespora spores were 0.6 and 12.4 spores/week, respectively, in the third week of November (Figure 2b). The time period lasting from the third week in October to the first week in November displayed accumulated rainfalls of 261.7 mm.

The variations in severity of the RCS disease displayed a high correlation with the fluctuations in populations of Coniella and Corynespora spores, as well as in the total of spores (Table 5). The correlation between the severity of the disease on the leaves of the lower section of the plant (evaluated in September) and the accumulated number of Coniella and Corynespora spores and the total of spores was positive and significant for both years. In the middle and top sections of the plant, the severity of the disease was evaluated in calyces and correlated positively with the populations of Corynespora spores and with the total of spores in the air.

Figure 2 Mean weekly severity of leaf (September) and calyx spotting (November) in roselle (RCS) estimated on a scale of 0 to 5, where 0=healthy and 5=severe damage to untreated plants in 2017 (a) and 2018 (b). Number of Coniella and Corynespora spores per week trapped in the field using a Burkard volumetric spore trap. Vertical bars represent standard errors. 

Discussion

The study on the dynamics of spores present in the air is important to understand the spatial and temporary distribution pattern of the pathogen’s inoculant, in order to propose integrated disease management strategies.

Identification of fungi. In this investigation, the genus Coniella (=Pilidiella) was the most frequently found in the plantation and the most abundant in the air, whereas Corynespora was less frequent in the plantation and in the samples taken in the air. Lasiodiplodia was even less frequent and abundant on the crop. In turn, the Curvularia and Alternaria spores were abundant in the air, and Curvularia has been associated with RCS (^{Trujillo-Tapia and Ramírez-Fuentes, 2015}). However, the study was centered on the genera Coniella and Corynespora, since they have been reported as pathogenic agents in roselle with the species P. (=Coniella) diplodiella as the cause of aqueous spots on leaves and defoliation of plants (^{Correa et al., 2011}), C. javanica as the cause of leaf and calyx blight and overall wilting of the plant (^{Barrón-Coronado et al., 2019}), and Corynespora cassiicola, reported as the causal agent of circular to irregularly-shaped, sunken necrotic spots on the calyx, and on leaves, circular to irregularly-shaped spots with light brown centers, black edges and purple rings that form necrotic areas when they join (^{Ortega-Acosta et al., 2015}). The pathogenicity tests performed with C. diplodiella and C. cassiicola confirmed a very similar symptomatology between both fungi.

Weather and pathogens. The Coniella and Corynespora spores were caught over the plantation during the entire production cycle, starting on day 10 after planting. The first rainfalls caused the germination of seeds left on the soil during the previous cycle, which allows for a good availability of host tissue. On the other hand, ^{Hernández-Morales et al. (2018}) mention that Corynespora cassiicola has other, alternate hosts such as weeds and plants sown in the production area, which may explain the levels of inoculants from the beginning of planting. However, the largest spore populations appeared during the development of flower buds and until harvesting in October and November. Flowers began emerging in late September and coincided with days with less than 12 hours of sunlight, which has been documented by ^{Muslihatinn and Daesusi (2014}). The concentration of spores in the air over the crop was widely related to the rainfall, temperature, wind speed and global radiation in the environment before their capture. However. Negative correlations were found between the rains and the concentration of Corynespora spores, seemingly during heavy rainfalls >138 mm in September 2017, when the density of spores in the air diminished. This behavior has been reported for other fungi, such as Botrytis cinerea, indicating that raindrops reduce the number of spores in the air (^{Blanco et al., 2006}). A similar situation was reported by ^{Pakpour et al. (2015}) in an urban study that indicated that the concentrations of spores in the air were higher when rainfall was less and temperatures were higher; fungal spores persist in the air during the dry seasons and rain can reduce the number of spores present in the air. In contrast, ^{Ganthaler and Mayr (2015}) indicate that the humid soil conditions and the leaves that remain after a rainfall contribute to an increase in the density of fungal spores in the air.

The maximum and average daily temperature presented a positive correlation with the concentration of Coniella spores. In this study, the period with the highest number of spores corresponded with periods of temperatures between 30-33.6°C, which does not coincide with reports for other fungi such as Botrytis cinerea by ^{Sosa-Alvarez et al. (1995}), who stated that a large number of spores are produced at a temperature near 15-22 °C after 7 days of continuous humidity. Despite this, the production and dispersal of spores are two different events, and it is possible that these maximum temperatures contributed to dispersion in the air, thus the greater capture, given that the minimum temperatures in the daytime of the aforementioned days were 19.4-20.4 °C (Table 3 and Figure 1). This situation may be explained by the results found with Botrytis cinerea, in which two phases were discerned: in the first, spores are released by the conidiophore with an intervention of a hygroscopic mechanism that controls the turgidity of the conodiophore, and secondly, there is an episode of conidial transport with the intervention of physical agents (wind and raindrops), which commonly takes place in the early hours of the morning, when the relative humidity chances and as temperatures rise (^{Blanco et al., 2006}). On the other hand, the Corynespora populations in this study displayed a significantly negative correlation with the minimum temperature. The largest populations appeared in temperatures between 17.2 and 20.7 °C, which differs from reports by ^{Kamei et al. (2018}), who indicate that C. cassiicola causes foliar spots to progress in tomato at an optimum temperature of 24 to 25 °C, along with a relative humidity of 80-85%. In turn, ^{Sharma (2017}) report a wider range of temperatures of 16-32 °C; longer-lasting humidity increases the number of lesions caused by foliar spots in cotton plants caused by C. cassiicola in all temperatures tested, of 16, 20, 24, 28 and 32 °C, and the largest lesions were displayed at 28 °C with leaf exposed to humidity for 48 hours. The spore distribution patterns in roselle canopies have rarely been studied. Further data and studies are required to fully understand the epidemiology of the roselle disease complex.

The presence of winds with maximum speeds of >4.4 km hr^-1 and the time with sunlight every day <12 hrs were negatively correlated with the low Corynespora spore population densities in the air. At the same time, average daily global solar radiation was >249 (W/m²), which favored the increase of Coniella inoculant in the air and the total of spores. The effect of sunlight on fungal sporulation has been documented and studied experimentally for some fungi; the main photomorphogenetic effect is through the induction of sporulation (^{Ensminger 1993}). However, the wavelengths that induce sporulation in low amounts may inhibit sporulation in higher amounts (^{Paul 2000}) and these same wavelengths have stimulating or inhibiting effects on the development of the conidia and affect fungal photomorphogenesis (^{Maddison and Manners 1973}).

The annual weather variation, mainly regarding temperature and, to a lower extent, rainfall, may determine the concentrations of spores in the air. The year with the lowest rainfall levels seems to have resulted in higher spore concentrations, which may have implications on the control of the disease and of climate change.

Correlation between the severity of RCS and Coniella and Corynespora spores. The results of the present investigation showed that the interaction between the host, climate and inoculant that prevailed during the phenological stages of the roselle crop favored the development of the disease.

In the present study, there were positive significant correlations between the severity of RCS and the concentration of Coniella and Corynespora spores and the total spores. The levels of Coniella inoculant had a higher relation with the severity in leaves, and on the other hand, the increase of Corynespora inoculant on the crop was related with the severity of the disease in the development of flowers. Both fungus genera have been reported as causal agents in roselle diseases: ^{Correa et al. (2011}) reported Pilidiella (=Coniella) diplodiella with symptoms of aqueous spotting of leaves; ^{Barrón-Coronado et al. (2019}) reported Coniella javanica, pathogen of leaf and calyx blight; ^{Ortega-Acosta et al. (2015}) reported Corynespora cassiicola as a pathogen of leaf and calyx spotting.

During the stage of vegetative growth, from seed germination to early flower budding, the presence of Coniella spore populations, and a continuous presence of susceptible tissues (young and mature leaves) from July to September are not a limitation in terms of availability of host tissue. However, in September, before flower emergence, an important defoliation was observed of leaves affected by foliar spots, along with a low concentration of inoculants in the air, due to the reduction of spores in the air due to heavy rainfalls in said month, although the ^{SMN (2019)} reports a normal rainfall in September of >434.8 mm. When the roselle plant flowers (October and November), it coincides with the accelerated growth phase of the disease in the young foliage with high inoculant levels (Coniella and Corynespora with >100 and 8 spores/week respectively over the crop) and with ideal weather conditions for the epidemic (average daily temperature 25.3-26.6 °C, average radiation of 235.9-317.1, with a relative humidity >84% and with 12-11.2 hrs of sunlight per day).

These results may have implications for the control of the disease, where up to seven applications of fungicides (mancozeb, chlorothalonil and benomyl alternating with copper oxychloride) have been reported, all programmed in pre-flowering, flowering and fruition, which curtail infections by Corynespora cassiicola, and therefore a lower severity of the disease and a higher fresh and dry weights of the calyces (^{Ortega-Acosta et al., 2019}). However, it is important to make an effort to reduce the applications of untimely and potentially unnecessary fungicides, with information on the sampling of airborne inoculants, as near as possible to real time, which helps estimate the potential risk of accumulation of spores and their germination, which would help adjust the intervals of application of fungicides.

Conclusions

Coniella and Corynespora spores were the common genera present in the air over the roselle plantation. The weather variations, mainly temperature and rainfall, determine the concentrations of spores over the plantation. The severity of the calyx spotting was positively and significantly correlated with the number of Coniella and Corynespora spores accumulated every week.