Molecular characterization of two papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander,Colombia

Papaya ringspot virus is an RNA virus that belongs to the genus Potyvirus, family Potyviridae and affects both papaya and cucurbits, causing great economic losses. PRSV isolates are divided into biotypes P and W; both biotypes naturally infect plants in the family Cucurbitaceae, whereas the P type also naturally infects papaya (Carica papaya L). In the present study, we report the full-length genome sequence of two PRSV-P isolates sampled from the Campo Hermoso (PRSV-CH) and Villa del Rosario (PRSV-VR) localities in Norte de Santander, Colombia. The genomes of these PRSV isolates are 10,326 nt in length and have a predicted ORF of 3344 aa. The identity among Colombian PRSV isolates is 96.9% and 97.3% at the nucleotide and deduced amino acid levels, respectively. PRSV isolates from China had the lowest identity at 78.3% and 89.2% (nucleotide-amino acid), whereas the highest identities were detected in PRSV isolates from Mexico, Venezuela and Hawaii. At the polyprotein level, the amino acid composition surrounding the active polyprotein cleavage sites differ in the Colombian PRSV sequences. The predicted cleavage site in P1/HC-Pro is LEQY/N – LEQY/S instead of MEQY/N. Both of the Colombian PRSV isolates have a putative recombination event in the P1 coding region, which is common in all PRSV isolates from the American continent. The new full-length PRSV sequences from Colombia provide a better understanding of the dynamics of papaya ringspot virus infections in papaya in Colombia and worldwide.     

The ability of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis>strains overcoming the transgenic resistance of papaya conferred by the coat protein gene is not correlated with higher degrees of sequence divergence from the transgene

The coat protein (CP) gene mediated transgenic resistance is found to be the best approach for protecting papaya plants against the destructive disease caused by Papaya ringspot viruses(PRSV). In order to study the variability of PRSV and the potential threat to the CP-transgenic resistance, five virus isolates were collected from transgenic plants of papaya line 16-0-1, which carry the CP gene of the typical mosaic strain of Taiwan PRSV YK, in an approved test field and fourteen from untransformed papaya plants in different areas of Taiwan. The results of biological, serological, and molecular characterization indicated that all isolates are related to PRSV YK. Among them, the isolate 5--19 from the transgenic line and the isolates CS and TD2 from untransformed papaya were able to overcome the YK CP gene-mediated resistance of papaya lines 18--2--4, 17-0-5, and 16-0-1, which provide high degrees of resistance to different geographic PRSV strains of Hawaii (HA), Mexico (MX), and Thailand (TH). These three isolates were also able to cause symptoms on untransformed papaya plants more severe than those induced by YK. In addition to the host reactions, the variability of the collected 19 isolates was also analyzed and compared with YK and other geographic strains by heteroduplex mobility assay (HMA) and sequence analyses. The results of HMA indicated that the CP genes of isolates 5--19 and TD2 are more divergent than those of other isolates when compared with YK. However, sequence analyses of the transgenic-resistance overcoming isolates 5-19, CS, and TD2 revealed that their CP coding regions and the 3 untranslated regions (UTRs) share nucleotide identities of 93.9–96.6% and 94.2–97.9% with those of YK, respectively; whereas the other geographic strains of HA, MX, and TH that could not overcome the transgenic resistance share lower nucleotide identities of 89.8–92.6% and 92.3–95.3% with those of YK, respectively. Our results indicate that the ability for overcoming the transgenic resistance is not solely correlated with higher degrees of sequence divergence from the transgene. The possible mechanism for overcoming the transgenic resistance and the potential threat of these PRSV strains to the application of the transgenic papaya lines carrying PRSV YK CP gene are discussed.     

Characterization of Colletotrichum truncatum from papaya,pepper and physic nut based on phylogeny,morphology and pathogenicity

Colletotrichum truncatum (syn. C. capsici) has been identified as the causal agent of anthracnose on various hosts, predominantly pepper (Capsicum spp.) plants. The aim of this study was to determine whether C. truncatum isolates infecting papaya, pepper and physic nut in southeastern Mexico are morphologically, genetically and pathogenically different, in order to improve disease management strategies. A total of 113 C. truncatum isolates collected from five producer states were subjected to phenotypic characterization and divided into six different morphological groups. These morphological traits and the location of the isolates were used to select a subset of 20 isolates for further studies. Differences in the pathogenicity of the isolates were tested with a cross‐inoculation assay using pepper, papaya and physic nut. The pathogenicity tests revealed that all isolates could infect the three hosts and produce typical anthracnose symptoms, indicating a lack of host specificity for this species and therefore its pathogenic potential on other plants. Phylogenetic analysis using internal transcribed spacer (ITS) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) sequences of the C.   truncatum isolates from this study and reference strains was performed, grouping the isolates into a monophyletic clade. This study reports for the first time the characterization of C. truncatum causing anthracnose disease on three different hosts in Mexico.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.     

Thiophanate-methyl sensitivity and fitness in Lasiodiplodia theobromae populations from papaya in Brazil

Stem-end rot, caused by Lasiodiplodia theobromae, is an important postharvest disease of papaya in Brazil. The use of fungicides is one of the main disease management measures. However, there are no data available on the sensitivity of L. theobromae to thiophanate methyl (methyl benzimidazole carbamate), the most common fungicide used in papaya orchards in northeastern Brazil. Thus, the effective concentration that results in 50 % of mycelial growth inhibition (EC₅₀) of 109 isolates, representing five populations of the pathogen was estimated in vitro. Seven components of fitness were measured for the 10 isolates with lower and high values of EC₅₀. Of the 109 isolates, 20.2 % were resistant to the fungicide with EC₅₀ values greater than 300 μg ml^?1, whereas the remaining 79.8 % were sensitive with an average EC₅₀ of 1.87 μg ml^?1. The EC₅₀ values for the resistant isolates were significantly (P?≤?0.05) higher than those for the sensitive isolates. When the fitness components were evaluated, only in relation to the spore production was significant difference among sensitive and resistant isolates, and resistant isolates showed sporulation capacity significantly lower than the S isolates, indicating a fitness cost.     

Importance of cucurbits in the epidemiology of Papaya ringspot virus type P

Papaya ringspot virus type P (PRSV‐P) systemically infects Carica papaya and species belonging to the family Cucurbitaceae. Attempts to recover PRSV‐P from naturally infected cucurbit plants grown near or among diseased papaya trees have shown conflicting results worldwide. This study aimed to evaluate the natural infection of cucurbit species grown among and near papaya trees infected with PRSV‐P in Brazil. Natural infection of cucurbits with PRSV‐P occurred in zucchini squash but not in watermelon and cucumber. However, several attempts to recover PRSV‐P from numerous Cucurbita pepo cv. Caserta (zucchini squash) plants grown 5–80 m from diseased papaya trees in the field failed. Mechanical inoculations of Cucurbita pepo cv. Caserta, Cucurbita maxima cv. Exposição (pumpkin), Cucumis sativus cv. Primepack Plus (cucumber) and Citrullus lanatus cv. Crimson Sweet (watermelon) with five Brazilian PRSV‐P isolates showed that zucchini squash was the most susceptible species followed by watermelon and cucumber, while pumpkin was not infected. The results confirmed the variable susceptibility of cucurbit species to experimental and natural PRSV‐P infection. Given these facts, the control of the disease through roguing should focus mainly on diseased papaya plants, as has been practised successfully in Brazil for many years, and on those cucurbits particularly known to be susceptible to natural infection with PRSV‐P.     

Distribution and genetic diversity among Aspergillus flavus isolates across three agro-ecologies essential for maize cultivation in Ghana

Fungal mycotoxins are important contaminants of agricultural commodities that pose serious concerns to producers, consumers and exporters. Aflatoxin is a carcinogenic metabolite produced by the fungi Aspergillus flavus and A. parasiticus. These fungi and other mould species living in the soil contaminate several crops including maize. This study was undertaken with the following objectives: (i) to assess the presence of A. flavus and other moulds in Ghanaian soil; (ii) to determine the distribution and density of A. flavus under three agro-ecologies noted for major maize production; and (iii) to assess the effect of percentage of carbon (C) and nitrogen (N) and soil pH on A. flavus population densities and diversity in the isolates found across the agro-ecologies. The data showed seven moulds that were common across the agro-ecologies. Significant differences (P < 0.05) were observed in the A. flavus density and distribution within and across these agro-ecologies. Fumesua soils recorded the highest levels of A. flavus (1.185 × 10³ cfu g⁻¹) while Akomadan recorded the least (9.76 × 10² cfu g⁻¹). Percentage available C, N and soil pH did not significantly influence A. flavus density. The A. flavus isolates identified in this study varied in genetic sequence within the aflatoxin gene cluster, but these differences were not distinguishable by origin.     

Influence of native endophytic bacteria on the growth and bacterial crown rot tolerance of papaya (Carica papaya)

The native plant microbiome is composed of diverse microbial communities that influence overall plant health, with some species known to promote plant growth and pathogen resistance. Here, we show the antibacterial and growth promoting activities of autoclaved culture metabolites (ACM) from native endophytic bacteria (NEB). These NEB were isolated from a papaya cultivar (var. Cariflora) that is tolerant to bacterial crown rot (BCR) caused by Erwinia mallotivora. In this cultivar, bacterial colonization in tissues recovering from the disease was observed before onset of tissue regeneration or ‘regrowth’. We further isolated and characterized these bacteria and were able to identify two culturable stem NEB related to plant endophytic genera Kosakonia sp. (ex. Enterobacter sp., isolate EBW), and to Sphingomonas sp. (isolate EBY). We also identified root NEB under genus Bacillus (isolates BN, BS, and BT). Inhibition assays indicated that ACM from these NEB promptly (within 18-30 h) and efficiently inhibited (60–65% reduction) E. mallotivora proliferation in vitro. When surface-sterilized papaya seeds were soaked in ACM from isolates EBY and EBW, germination was variably retarded (20–60% reduction) depending on plant genotype, but plant biomass accumulation was significantly stimulated, at around two-fold increase. Moreover, greenhouse experiments show that ACM from all isolates, especially isolate EBW, significantly reduced BCR incidence and severity in a susceptible genotype (var. Solo), at around two-fold. In general, our observations of pathogen antagonism and plant growth promotion leading to disease reduction, suggested the influence of native endophytic bacteria to increased fitness in plants, and tolerance against the re-emerging crown rot disease of papaya.

     

Morphological, pathological and genetic diversity of Colletotrichum species responsible for anthracnose in papaya (Carica papaya L)

Recently, anthracnose has become a major problem in papaya production and postharvest stages. The occurrence of both Colletotrichum gloeosporioides and Colletotrichum capsici has been demonstrated in this crop. The differential response of these pathogens to fungicides has highlighted the need to use rapid and accurate techniques to identify them. Thus, the objective of this study was to reveal the genetic diversity of Colletotrichum isolates in Mexican papaya fields. C. gloeosporioides-and C. capsici-specific primers were successfully used to detect the pathogens from different papaya parts. A combination of morphological characters, molecular techniques and pathogenicity tests were used to characterize 37 isolates from different localities of five papaya-producing states. Analyses of the 5.8-ITS region and arbitrarily primed-PCR revealed intraspecific groups; most of the isolates within these groups have the same geographical location and morphological characteristics. Knowledge of the genetic diversity of Colletotrichum spp. in Mexican papaya fields will facilitate the identification of the pathogen population in this crop in order to select the appropriate fungicide to control anthracnose, as well as to improve genetic resistance breeding programs.     

Comparison between the penetration characteristics of methyl bromide and ethanedinitrile through the bark of pine (Pinus radiata D.Don) logs

BACKGROUND

Ethanedinitrile (EDN) is a fumigant being commercialized worldwide as an alternative phytosanitary treatment to methyl bromide (MB) for forest products. The penetration characteristics of MB and EDN were measured through the bark of wooden blocks (100 × 100 × 50 mm) cut from the upper (average bark thickness 5 ± 2 mm) and lower (average bark thickness 25 ± 5 mm) trunk of recently felled pine (Pinus radiata D.Don) trees. Doses of 48 g m⁻³ MB and 50 g m⁻³ EDN were applied to chambers at 10 and 20°C for 10 h.

RESULTS

Penetration of MB was influenced by the interaction between fumigation time and temperature, with concentrations increasing at a higher rate at 20°C compared with at 10°C. After 10 h, an average concentration of 8.05 ± 0.89 g m⁻³ had penetrated the bark of log sections at 20°C, whereas 5.20 ± 0.89 g m⁻³ was measured at 10°C. By contrast, the factors examined in this study did not significantly impact the penetration of EDN. Concentration × time (CT) values for MB under the bark were 35.20 ± 2.30 g h m⁻³ at 10°C and 55.85 ± 9.58 g h m⁻³ at 20°C; whereas for EDN, CT values were 19.50 ± 6.80 g h m⁻³ at 10°C and 19.08 ± 4.10 g h m⁻³ at 20°C.

CONCLUSION

MB can achieve a higher concentration under the bark of log sections during simulated fumigations, but all of the factors examined affected the ability of MB to penetrate the bark of wooden blocks. By comparison, the penetration of EDN through the bark is more consistent than MB under laboratory conditions. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Inter-individual genetic variation in the temperature response of Leptosphaeria species pathogenic on oilseed rape

It is important to understand the likely response of plant pathogens to increased temperatures due to anthropogenic climate change. This includes evolutionary change due to selection on genetically based variation in growth rate with temperature. We attempted to quantify this in two ways. First, radial mycelial growth rates in agar culture were determined for a collection of 44 English isolates of Leptosphaeria maculans and 17 isolates of L. biglobosa, at 14 temperatures. For L. maculans the genotypic variances in four parameters were measured: minimum temperature allowing growth, optimum temperature, growth rate at the optimum temperature, and growth rate at the highest usable temperature, 31.8°C. The standard deviations were 0.068°C, 1.28°C, 0.21 mm/day, and 0.31 mm⋅day⁻¹⋅°C⁻¹, respectively. For L. biglobosa, these figures were, respectively: immeasurably small, 1.31°C, 0.053 mm/day, and 0.53 mm⋅day⁻¹⋅°C⁻¹. In addition, the incidence and severity of phoma stem canker in planta over a natural growing cycle at four temperatures (16, 20, 24, and 28°C) around the average culture optimum were determined. There was no correlation between in vitro and in planta growth, and the decrease in pathogen measures either side of the optimum temperature was much less for in planta growth than for in vitro growth. We conclude that both pathogens have the capacity to evolve to adapt to changes in environmental conditions, but that predictions of the effect of this adaptation, or estimates of heritability in natural conditions, cannot be made from measurements in vitro.     

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation

Fusarium mangiferae is a major causal agent of mango malformation disease (MMD) worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for quantitative detection of F. mangiferae in China. The LAMP primer set was designed based on a RAPD marker sequence and positive products were amplified only from F. mangiferae isolates, but not from any other species tested, showing a high specificity of the primer sets. The detection limit was approximately 2.26 × 10⁻⁴ ng/μl plasmid DNA when mixed with extracted mango DNA. Quantification of the pathogen DNA of MMD in naturally collected samples was no significant difference compared to classic real-time PCR Additionally, RealAmp assay was visual with an improved closed-tube visual detection system making the assay more convenient in diagnostics.     

Genetic diversity and population structure of Lasiodiplodia theobromae from different hosts in northeastern Brazil and Mexico

Lasiodiplodia theobromae is one of the most frequent fungal pathogens associated with dieback, gummosis, leaf spot, stem-end rot and fruit rot symptoms in cashew, mango, papaya and grapevine. In this study, the variation in the genetic diversity of 117 L. theobromae isolates from northeastern Brazil (n = 100) and Mexico (n = 17), which were collected from these four crops, was analysed using microsatellite markers. The results revealed low genetic diversity among L. theobromae populations and the existence of two genetic groups. All Mexican isolates were grouped with Brazilian isolates, suggesting a low level of differentiation between these populations. Furthermore, no evident host or climate-based population differentiation was observed for L. theobromae in Brazil. The populations studied were mostly clonal, but additional studies are needed to better understand the mode of reproduction of the pathogen. The low genetic diversity of L. theobromae populations in northeastern Brazil suggests that resistant cultivars could be used as a durable management strategy to reduce the impact of the diseases caused by this pathogen.     

Isolation of antagonistic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. against a potato scab pathogen from a field cultivated with wild oat

Fungi, actinomycetes, and bacteria isolated from soil and plant samples from a potato field in which wild oat (Avena strigosa) had been pre-cultivated were screened for microorganisms that can be used as biocontrol agents for common scab of potato. Of 342 isolates assessed in initial pot trials for their suppressive effect on the severity of potato scab caused by Streptomyces turgidiscabies, 26 isolates were selected as antagonistic candidates based on their ability to reduce disease severity, then tested in a second pot trial. Of the 26, five actinomycetes, isolated from either the rhizosphere soil of wild oat or the soil adhering to potato stolons and tubers, were selected as antagonists. A comparison of partial sequences of 16S rRNA genes from the five isolates indicated that they belong to the genus Streptomyces. Of these five, WoRs-501 most strongly inhibited in vitro mycelial growth of S. turgidiscabies and was also the most effective in suppressing potato scab in a third field pot trial. In that pot trial, a 10% (v/v) mix of WoRs-501 (6.2 × 10⁸ colony-forming units [CFU]/g dry mass) decreased the disease severity by 78–94% in comparison with the untreated control at 5 × 10⁴ to 5 × 10⁶ CFU S. turgidiscabies/g dry soil. WoRs-501 also grew well in vitro at a wide range of pH levels and temperatures. These results suggest that WoRs-501 is a promising candidate for biocontrol of potato scab.     

Genetic analysis of an attenuated <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> strain applied for cross-protection

Papaya ringspot virus (PRSV) HA 5-1, a nitrous acid-induced mild mutant of severe strain HA, widely applied for control of PRSV by cross-protection, was used to study the genetic basis of attenuation. Using infectious clones, a series of recombinants was generated between HA 5-1 and HA and their infectivity was analyzed on the systemic host papaya and the local lesion host Chenopodium quinoa. The recombinants that contained mutations in P1 and HC-Pro genes caused attenuated infection on papaya without conspicuous symptoms, similar to HA 5-1. The recombination and sequence analyses strongly implicated two amino acid changes in the C-terminal region of P1 and two in HC-Pro of HA 5-1 involved in the attenuated infection on papaya. The recombinants that infected C. quinoa plants without local lesions contained the same mutations in the C-terminal region of HC-Pro for attenuated infection on papaya. We conclude that both P1 and HC-Pro bear important pathogenicity determinants for the infection on the systemic host papaya and that the mutations in HC-Pro affecting pathogenicity on papaya are also responsible for the inability to induce hypersensitive reaction on C. quinoa.