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Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

Leaf rust caused by Cerotelium fici (Cast.) Arth. is the main disease affecting Moraceae family plants, such as Ficus and Morus species (Galleti and Rezende 2016; Srikantaswamy et al. 2006). In August 2020, rust symptoms were observed in 100% of mulberry (Morus nigra L.) trees in an experimental orchard (Piracicaba, SP, Brazil; 22°42’28”S, 47°37’42”W). Mulberry leaves with high rust severity became yellowish and fell-off prematurely. Pustules were light brown with yellowish halo and presented mean size of 0.9 mm2. Uredinial paraphyses (n = 50) measured 42.2 ± 0.67 µm long with wall uniformly ca 0.6-1.1 μm thick. Urediniospores were brownish, echinulate, globoid to broadly ellipsoid, and measured 27.1 ± 0.29 × 21.0 ± 0.27 µm with a wall thickness of 0.6 ± 0.01 µm (n = 100). The morphology of the urediniospores observed in this study was similar to that reported in the literature for C. fici on Morus alba and Ficus spp. (Gupta et al. 1994; McKenzie 1986; Hennen et al. 2005). We used a low-coverage genome-skimming approach to retrieve genetic information of the rRNA cluster and the mtDNA. Genomic DNA was extracted from 3-4 mg of stored urediniospores at -80 °C, macerated in liquid nitrogen, using a modified cetyl trimethylammonium bromide extraction procedure (Lo Piccolo et al. 2012), and sequenced with 150-bp paired-end reads on Illumina NovaSeq 6000 System. Raw data, (45,761,957 X 2 reads) were assembled with SPAdes v3.15.1 (Bankevich et al., 2012) and the output used to create a custom BLAST database. Loci used for the phylogenetic analyses were identified by BLASTn using, as a query, sequences of C. fici from Ficus sp. from Australia publicly available: Accession No. MH047210.1 for the rRNA and MW036502.1 for COX3. The retrieved sequences were deposited in GenBank under accession numbers OM296992 and OP797407 for the partial rRNA cluster and COX3, respectively. The Bayesian inference phylogenetic analysis of the three concatenate loci (18S, 28S, and COX3) revealed that the isolate obtained in this study (MN1) was clustered in a well-supported clade with C. fici type species. Pathogenicity tests were conducted using mulberry potted plants under greenhouse conditions (25 ± 5 °C). The urediniospores suspension (5 × 104 urediniospores ml-1) with 0.05% Tween 20 was sprayed with an airbrush on fully expanded leaves until run-off. As a control, mulberry plants were sprayed with distilled water and kept under the same conditions. Inoculated and mock-inoculated plants were kept in a dark moist chamber at 23 °C (± 2 °C) for 24 h. After this period, plants were moved to the greenhouse. The experimental design was completely randomized with five replicates, each replicate consisted of one potted plant and the experiment was performed twice. At 12 days post-inoculation, all inoculated plants showed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. The first symptoms were small pustules on the abaxial surface of fully expanded leaves. Small chlorotic lesions were observed on the adaxial leaf surface, which evolved into necrotic lesions. The pathogen was re-inoculated into potted plants, where it was maintained through monthly inoculations. To our knowledge, this is the first report of mulberry rust on M. nigra in Brazil. As mulberry leaves are the only natural food for silkworm (Bombyx mori L.), rust poses a significant threat to the sericulture industry because the disease can decrease production and quality of mulberry foliage.

Field resistance to quinone outside inhibitor (QoI) fungicides in Alternaria alternata causing Alternaria brown spot of mandarins was reported by growers in Brazil in 2017. Conidial germination tests were performed, and isolates showed effective concentration to inhibit 50% of conidia germination (EC50) to be over 100 ppm. This is the first report of QoI resistance in A. alternata causing Alternaria brown spot in the state of São Paulo, Brazil.

Citrus black spot (CBS), caused by Guignardia citricarpa, is the most important fungal disease of orange trees in Brazil. The spatial pattern of CBS-symptomatic trees was evaluated using the binomial dispersion index (D), Ripley's K function (K), and a Monte Carlo test for minimum mean distance (d) to understand the distribution of the pathogen. Disease was monitored in 7,790 citrus trees from four commercial groves. In one grove, disease incidence was assessed from 1999 to 2001 and, in the others, disease assessments were conducted only in 2002. Infected trees were aggregated based on the three statistical analyses used (D, K, and d) regardless of the CBS incidence. The binomial index of dispersion (D) indicated aggregation of CBS-affected trees for all groves and for various quadrat sizes (2 by 2, 3 by 3, 4 by 4… up to 10 by 10). According to Ripley's K function, the dependence among symptomatic trees comprised two to three neighboring trees. Disease dispersion occurred at distances below 24.7 m according to the test for d. This suggests that the dispersion of inoculum is highly important over short distances. As a consequence, the required sample size to achieve a level of accuracy of C = 20% increases exponentially with the decrease in incidence of CBS below 15% infected plants.

Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylor's power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.

A simulator for the enlargement of cohorts of circular lesions on cohorts of host tissue was used to examine five epidemiological parameters: radial rate (mm day-1) of lesion expansion, k exp; maximum basic infection rate, R m; proportion of lesion area as infectious, f; initial lesion size (mm2), z; and proportion of susceptible host sites, s. Based on the proportion of disease severity at day 50 and the proportion of the total disease that originated solely from lesion expansion, kexp was the most sensitive of the five parameters. A radial rate of only 0.1 mm day-1 resulted in a proportion of >0.7 of the diseased area that came from lesion expansion. In an extensive survey of phytopathological literature, many plant pathogens had radial rates greater than 0.1 mm day-1, which would result in a proportion of >0.95 of the total disease that comes from lesion expansion. Susceptible host sites, s, was a sensitive parameter, as this determined the host area into which lesions could expand. Naturally, Rm was a sensitive parameter for the proportion of disease on day 50, as it controlled the overall speed of the epidemic. Initial lesion size was a relatively insensitive parameter, although z interacted significantly with s. The greatest proportion of disease that originated from lesion expansion occurred with fast kexp, small z, and low values of s, Rm, and f. The model was validated with lesion numbers and severities obtained in natural epidemics of Cercospora medicaginis on alfalfa and Exserohilum turcicum on maize. We recommend that the ‘epidemic quintuplet’ used to describe polycyclic epidemics be expanded to the ‘epidemic sextuplet’ with the inclusion of kexp, since lesion expansion is a major component of many polycyclic epidemics.

A simulation model was developed for bean rust epidemics based on infections that occurred in daily cohorts of leaf growth. The most sensitive function in the model was the daily correction of the basic infection rate for the amount of healthy host tissue on each cohort that was available for infection. Components of the basic infection equation, i.e., maximum basic infection rate (R max ) and parameters for environmental favorability (F) and the rate of the decreased susceptibility of leaves with age (A), also were sensitive factors. A greater pustular area on day 70 occurred when the rate of host growth was slower than standard, and a lower pustular area occurred when the rate of host growth was faster than standard. The maximum leaf area per plant was a relatively insensitive parameter. The total pustular area (y 70 ) was greatest when an epidemic was initiated at the highest level (f 0 = 0.01) of initial infection, on the earliest day of infection [i(f 0 ) = 2], and at maximum environmental favorability (F = 1.0). With epidemic rates (k G ) calculated with the Gompertz transformation, the fastest rates also occurred at f 0 = 0.01, i(f 0 ) = 2, and F = 1.0. In contrast, with epidemic rates (r) calculated with the logistic transformation, the fastest rates occurred with epidemics begun at the lowest level (f 0 = 0.00001) of initial infection. The logistic transformation of the low levels of y 50-70 caused inflated values of r. The model was verified by simulating an epidemic using temperatures and hours of leaf wetness that were recorded during an actual season. The curve for the increase in pustular area simulated by the model was representative of the progress of rust observed in the natural epidemic.

The FERRUGEM simulation model of bean rust epidemics, based on infections of Uromyces appendiculatus that occur on daily cohorts of bean leaf growth, was validated with data from 35 bean rust epidemics monitored in the southeastern region of Brazil. Subjective and goodness-of-fit tests of data were used. Good agreement was obtained between model output and real data in the majority of epidemics. Linear regression lines of Gompertz-transformed proportions of pustular area predicted by the model did not differ for both slope and intercept from regression lines of observed pustular areas in 60.0% of the epidemics. In 71.4% of the epidemics, there was no significant difference in intercepts. Lack of agreement between model-predicted outcomes and observed epidemics occurred in two situations : when the initial proportion of pustular area observed in the field was very low (