Necrotic Symptoms Research Articles

Prevalence of the Potato virus Y strain composition impacting Michigan seed potato production

Potato virus Y (PVY) is a complex of multiple strains that cause a variety of foliar and tuber symptoms. Several current strains have potential to result in variety-dependent yield and quality losses. Understanding the local PVY strain population and variety responses are critical for detection and management. From 2019-2023, PVY strain composition was monitored across seven Michigan seed potato-producing counties growing prevalent chip, russet, and tablestock market classes. Using multiplex reverse-transcriptase polymerase chain reaction methods, PVY strains were identified from potato tuber or leaf samples collected through the certification program (N = 78 positive lots). PVYN-Wi remained most prevalent in Michigan, however, tuber necrotic strains PVYNTN and PVYE also continued to be detected. These, as well as some PVYN-Wi isolates, are known to cause necrotic symptoms in tubers, causing yield and quality reductions for susceptible potato cultivars. As in other U.S. potato-growing regions, PVYN-Wi and PVYNTN were most prevalent in Michigan seed potato lots. Of note, mild to severe tuber necrosis was observed in diagnostic samples positive with local PVYN-Wi strains. Parental strain PVYO appeared to re-emerge in 2023 and should be considered in certification and management. Mild or asymptomatic reactions may still lead to yield or quality losses and may pose a risk to more susceptible cultivars. Due to the diversity of strains and varieties prevalent in the Great Lakes region, continued uniform certification is recommended. Information gathered from this and future survey efforts will be valuable for growers, inspectors, and future breeding and selection efforts.

In vitro and in vivo Evaluations of the Antifungal Activity of Salicylic Acid and Silicon Against Ganoderma Boninense

Basal stem rot disease (BSR) in oil palms is one of the primary diseases that has led to the wide use of fungicides. This increased the development of fungal isolates resistant to fungicides and led to the search for alternative strategies to replace the use of fungicides. This study aimed to evaluate in vitro antifungal activity of salicylic acid (SA) and silicon (Si) in inhibiting mycelial growth of Ganoderma boninense using Poison Food Technique and to evaluate the in vivo efficacy of Si treatment on oil palm seedlings growth and resistance towards G. boninense. Percentage inhibition of radial mycelial growth (PIRG) was assessed, and Si treatment significantly reduced mycelial radial growth of G. boninenseup to 100% inhibition at concentrations of 200 and 250 mg/L. The half-maximal effective concentration (EC50) for Si was 68.57 mg/L, while for SiO2, it was 273.95 mg/L. The EC50 for salicylic acid was 381.33 mg/L. For in vivo evaluation, oil palm seedlings treated with Si at 150, 200, and 250 mg/L showed the lowest severity of leaf chlorosis and necrotic symptoms, which were 7.36%, 6.49%, and 4.05%, respectively. In contrast, the seedlings without Si showed the highest severity of leaf symptoms. Examination of internal bole tissues of oil palm seedlings treated with Si at a concentration of 250 mg/L also recorded a 3.0% mean percentage of disease severity compared to the untreated infected seedlings, which showed 35.0% disease severity. Our findings demonstrated the potential of Si application in controlling the BSR disease caused by Ganoderma boninense.

A new host record of Alternaria argyroxiphii on Upland cotton (Gossypium hirsutum)

For the first time, we isolated and identified Alternaria argyroxiphii on Upland cotton (Gossypium hirsutum) in Australia and elsewhere. A single-spored A. argyroxiphii isolate was obtained from diseased cotton leaves that exhibited small circular to enlarged and coalesced irregular necroses in the 2021–2022 cropping season in northwest New South Wales, Australia. We identified A. argyroxiphii based on its large conidia with filiform beaks and sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL). These multigene sequences of the cotton-A. argyroxiphii were 100% identical to those of the type A. argyroxiphii isolate CBS 117222; thus, confirming the identity. On two-true-leaf cotton seedlings, A. argyroxiphii was able to incite necrotic symptoms resembling those observed in the field, and was reisolated and re-identified, thus, fulfilling Koch's postulates. This is the first record of A. argyroxiphii on Upland cotton.

Genomic Analysis of a Novel Torradovirus "Rehmannia Torradovirus Virus": Two Distinct Variants Infecting Rehmannia glutinosa.

Rehmannia glutinosa, a crucial medicinal plant native to China, is extensively cultivated across East Asia. We used high-throughput sequencing to identify viruses infecting R. glutinosa with mosaic, leaf yellowing, and necrotic symptoms. A novel Torradovirus, which we tentatively named "Rehmannia torradovirus virus" (ReTV), was identified. The complete sequences were obtained through reverse-transcription polymerase chain reaction (RT-PCR), 5' and 3' rapid amplification of cDNA ends, and Sanger sequencing. The amino acid sequence alignment between the ReTV-52 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 51.3-73.3% and 37.1-68.1%, respectively. Meanwhile, the amino acid sequence alignment between the ReTV-8 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 52.7-72.8% and 36.8-67.5%, respectively. The sequence analysis classified ten ReTV strains into two variants. The ReTV-52 genome has two RNA segments of 6939 and 4569 nucleotides, while that of ReTV-8 consists of two RNA segments containing 6889 and 4662 nucleotides. Sequence comparisons and phylogenetic analysis showed ReTV strains clustered within the Torradovirus, exhibiting the closet relation to the squash chlorotic leaf spot virus. The RT-PCR results showed a 100% ReTV detection rate in all 60 R. glutinosa samples. Therefore, ReTV should be classified as a novel Torradovirus species. ReTV is potentially dangerous to R. glutinosa, and necessitating monitoring this virus in the field.

Orthotospovirus iridimaculaflavi (iris yellow spot virus): An emerging threat to onion cultivation and its transmission by Thrips tabaci in India

The yellow spot disease caused by the virus species Orthotospovirus iridimaculaflavi (Iris yellow spot virus-IYSV), belonging to the genus Orthotospovirus, the family Tospoviridae, order Bunyavirales and transmitted by Thrips tabaci Lindeman. At present, emerging as a major threat in onion (Allium cepa) in Tamil Nadu, India. The yellow spot disease incidence was found to be 53–73 % in six districts out of eight major onion-growing districts surveyed in Tamil Nadu during 2021–2023. Among the onion cultivars surveyed, the cultivar CO 5 was the most susceptible to IYSV. The population of thrips was nearly 5–9/plant during vegetative and flowering stages. The thrips infestation was 34–60 %. The tospovirus involved was confirmed as IYSV through DAS-ELISA, followed by molecular confirmation through RT-PCR using the nucleocapsid (N) gene. The predominant thrips species present in onion crops throughout the growing seasons was confirmed as Thrips tabaci based on the nucleotide sequence of the MtCOI gene. The mechanical inoculation of IYSV in different hosts viz., Vigna unguiculata, Gomphrena globosa, Chenopodium amaranticolor, Chenopodium quinoa and Nicotiana benthamiana resulted in chlorotic and necrotic lesion symptoms. The electron microscopic studies with partially purified sap from onion lesions revealed the presence of spherical to pleomorphic particles measuring 100–230 nm diameter. The transmission of IYSV was successful with viruliferous adult Thrips tabaci in cowpea (Cv. CO7), which matured from 1st instar larva fed on infected cowpea leaves (24 h AAP). Small brown necrotic symptoms were produced on inoculated plants after an interval of four weeks. The settling preference of non-viruliferous and viruliferous T. tabaci towards healthy and infected onion leaves resulted in the increased preference of non-viruliferous thrips towards infected (onion-61.33 % and viruliferous thrips towards healthy onion leaves (75.33 %). The study isolates shared 99–100 % identity at a nucleotide and amino acid level with Indian isolates of IYSV in the N gene. The multiple alignment of the amino acid sequence of the N gene of IYSV isolates collected from different locations and IYSV isolates from the database revealed amino acid substitution in the isolate ITPR4. All the IYSV isolates from India exhibited characteristic amino acid substitution of serine at the 6th position in the place of threonine in the isolates from Australia, Japan and USA. The phylogenetic analysis revealed the monophyletic origin of the IYSV isolates in India.

Cotylenchus cleo gen. n., sp. n., a new plant-parasitic nematode (Tylenchida: Anguinidae) parasitising on leaves of western sword fern plants from rainforests in Washington State, USA

Summary A new genus and species of anguinid nematode, Cotylenchus cleo gen. n., sp. n., was recovered from leaves of the western sword fern, Polystichum munitum, growing in rainforests in several locations of Olympic National Park, Washington State, USA. This new anguinid nematode induced distinct necrotic and chlorotic symptoms on fern leaves characterised by yellowing, light to brown areas between veins. The new species is characterised by a long and slender body, six incisures in the lateral field, robust stylet (12.0-13.5 μm) with large and rounded knobs, pyriform to elongate and abutting basal pharyngeal bulb, and long conical tail with pointed terminus; females having posteriorly located vulva (V = 74-80%) and well-developed post-vulval uterine sac (26-59 μm) and males having spicules 20-23 μm long. Phylogenetic analysis of the partial 18S rRNA and the D2-D3 expansion segments of the 28S rRNA genes showed that this anguinid nematode formed a separate evolutionary lineage different from all other Anguinidae taxa. The new species was also characterised by sequencing the ITS rRNA and COI genes.

Molecular characterization of emerging distinct watermelon bud necrosis virus (Orthotospovirus citrullonecrosis) variant causing necrosis disease on tomato in India

Watermelon bud necrosis virus (WBNV) cause serious economic impact on the cultivation of vegetable crops in the Indian subcontinent. In this study, an Orthotospovirus that infect tomato plants with typical severe necrotic symptoms had been identified as WBNV based on N gene sequences. The complete genome sequence of WBNV isolate (22ToNV) was determined and analyzed. The nucleotide sequences of the viral RNA segments in 22ToNV exhibit <91% similarity compared to the published WBNV genome sequence. Whereas, the amino acid sequences encoded by these segments, shared identity of about 96–98.54% among the other WBNV isolates. There is a notable divergence observed specifically within the intergenic regions (IGRs) of the S and M RNAs, with a lower similarity range of 78–84%. Nucleotide sequence comparisons and phylogenetic analysis showed that 22ToNV is a unique WBNV isolate having distinct genetic differences with other known WBNV isolates reported in different geographic regions of the country.

First Report of Penicillium oxalicum Causing Leaf Blight on 'Hongyang' Kiwifruit in China.

In September 2022, leaf blight symptoms (Fig. 1) were detected on six-year-old kiwi trees (Actinidia chinensis cv. 'Hongyang') in Xuzhou municipality (117.29º E, 34.23º N), Jiangsu Province. Early-stage disease symptoms included light brown necrotic lesions of irregular shape ranging in length from 0.2 to 2.4 cm, which turned into leaf blight after approximately 2 weeks. Those symptoms were similar to those previously reported during a Pestalotiopsis sp. infection on kiwi trees in Turkey (Karakaya 2001). Approximately 20% of the leaves from 300 trees examined in one kiwi orchard, 3000 m2 in size, showed the disease symptoms. Ten leading edges of symptomatic leaves were sterilized with 2% sodium hypochlorite for 1 min, rinsed twice with sterile ddH2O and cultured at 26ºC for 3 days on PDA medium containing 50 µg/ml chloramphenicol. The fungal colonies were collected, and the single spore isolation method was used to obtain four isolates. The obtained isolates showed white aerial mycelia that turned greyish after 2 days of cultivation on PDA medium at 26ºC. ITS (OR054113, OR054153-OR054155), TUB2 (OR060951-OR060953, OR249978), and CMD (OR255947-OR255950) genes were amplified using the ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively (Visagie et al. 2014a). The obtained ITS, TUB2, and CMD sequences shared 99.81%-100%, 96.72%-96.96%, and 90.17%-92.58% homology compared to the ex-type strain P. oxalicum CBS 219.30 (MH855125, KF296462, and KF296367), while the obtained ITS and TUB2 sequences showed 99.62%-99.81%, and 96.46%-96.72% identity compared to the representative strain P. oxalicum DTO 179B9 (KJ775647 and KJ775140) (Visagie et al. 2014b). The sequences obtained also showed high homology compared to P. oxalicum HP7-1 (ITS: 99.81%-100% homology; TUB2: 98.98%-99.38% homology; CMD: 94.71%-95.10% homology) (Li et al. 2022). A molecular phylogenetic tree was constructed using MEGA X with representative Penicillium strains retrieved from GenBank (Fig. 2). Microscope observations revealed the presence of curved septate hyphae. Conidia were colorless, unicellular, and ellipsoidal (5-8 μm in length; > 2000 observations), whereas conidiophores were mainly monoverticillate (approximately 20% of the conidiophores were biverticillate) (50-70 μm in length; 43 observations) and contained cylindrical phialides (13-15 μm in length). These findings are consistent with P. oxalicum morphology (Wu et al. 2022; Zheng et al. 2023). The pathogenicity of the four isolates was screened using healthy non-detached 'Hongyang' kiwi leaves. Fifteen leaves from five different two-month-old trees were used for each isolate, with three repetitions. For inoculation, a 10 mL solution containing 1 × 106 spores/mL was sprayed on the leaves. Sterilized water was used in the control experiment, which was carried out using fifteen leaves from five different two-month-old trees, with three repetitions. Inoculated trees were stored at 26ºC and 60% relative humidity for 2 days. All the infected leaves had necrotic lesions and leaf blight symptoms comparable to those found in the field, but the control leaves had no lesions. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphology analysis, fulfilling Koch's postulates. P. oxalicum is a common cause of blue mould in postharvest fruits (Tang et al. 2020). P. oxalicum has been recently reported as the causal agent of leaf spot in pineapple (Wu et al. 2022; Zheng et al. 2023), and leaf blight on maize (Han et al. 2023). Although Alternaria sp., Glomerella cingulate, Pestalotiopsis sp., Phomopsis sp., and Phoma sp. were previously isolated from kiwi leaves with blight symptoms (Kim et al. 2017), this is the first report of P. oxalicum causing leaf blight on kiwi trees worldwide. P. oxalicum is a well-known source of mycotoxins, such as secalonic acid (Otero et al. 2020), indicating that its presence in kiwifruit orchards may pose a significant risk to human health. The discovery of this hazardous pathogen in kiwi trees must drive the development of management strategies. Kiwifruit is an important dietary source of vitamins, fiber, folate, and potassium, and China is the major producer of kiwifruit, with more than 1.2 million metric tons harvested in 2021. This report will help to generate a better understanding of the pathogens affecting kiwifruit orchards in China.

First Report of Colletotrichum tropicale Causing Anthracnose on Pitaya (Hylocereus costaricensis) in Brazil.

Pitaya, Hylocereus costaricensis, is a species of the Cactaceae family and originated in the Americas (Ortiz & Livera, 1995). It has been cultivated in Brazil and has shown a great potential for fruit production and is currently present in several markets (Faleiro et al. 2021). In July 2018, infected plants of pitaya with symptoms of anthracnose were obtained from an orchard in Fortaleza, Ceará Brazil, (3°44'24.5"S 38°34'30.8"W), with 50% disease incidence. The symptoms observed consisted of well-defined and depressed stains, that initially appeared as reddish-orange spots and were surrounded by a border of dark-brown color. As the lesion progressed, the center became light brown or whitish in color, with black dots appearing later. Four cladodes were collected with anthracnose symptoms. The pathogen was isolated from symptomatic cladodes, which were surface disinfected with 1% v/v NaClO and 70% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA) medium and incubated under a light/dark (12h/12h) photoperiod. Two isolates were recovered from the lesions on cladodes. Pure cultures were obtained from single conidia produced on colonies grown on PDA medium, using an inoculation needle under a microscope. Colonies on PDA exhibited white aerial mycelia with an orange conidial mass. The colonies were light grey in the front and light orange in the reverse of the plate. Morphological features suggested that the isolates had the same characteristics as previously described for Colletotrichum spp. (Weir et al., 2012). In order to identify the species of the isolates, the genomic DNA of UFCM 0684 and UFCM 0685 isolates was extracted using the CTAB method and the ITS region, TUB2, ACT, GS, GAPDH gene fragments were amplified. PCR products were sequenced and the resulting sequences were submitted to phylogenetic analyses based on maximum likelihood for the combination of the genes. The isolates grouped within Colletotrichum tropicale with 99% bootstrap support. The sequences obtained in this study were deposited in GenBank as ACT (accession no. OL799311, OL799312), TUB2 (OL799313; OL799314), GAPDH (OL799315, OL799316), GS (OL799317; OL799318) and ITS (OL799319; OL799320). After that, the UFCM 0685 isolate was selected to study for further characterization. Conidia (n = 50) were 13.7 (length) × 4.7 μm (width) in average, hyaline, aseptate and cylindrical. To complete Koch's postulates, pathogenicity tests were performed in moist chamber for one week at 25°C with 80% relative humidity on a 12 h fluorescent light/dark photoperiod. The cladodes were wounded using a sterilized needle and inoculated with 10 µl of a conidial suspension (1 × 106 conidia/ml) on three cladodes with five wounds each. The same number of uninoculated cladode was used as control. The experiment was performed twice. Two weeks later, all inoculated cladodes showed necrotic symptoms, which were similar to the symptoms previously observed in the field. The uninoculated cladode remained symptomless. The fungus was reisolated from the inoculated cladode and its morphological characteristics were similar to the original isolate. Colletotrichum tropicale has been reported to cause anthracnose on H. costaricensis in Mexico (Nunez-Garcia et al. 2023), H. undatus, H. monocanthus and H. megalanthus (Evallo et al. 2022). For the best of our knowledge, this is the first report of anthracnose caused by C. tropicale in H. costaricensis in Brazil.

Overexpression of an NLP protein family member increases virulence of Verticillium dahliae

AbstractVerticillium dahliae is a xylem‐invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1‐Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild‐type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP‐overexpressing transformant was 22 times higher than that in plants infected with the wild‐type isolate, further supporting the critical role of VdNEP in infection. A VdNEP‐EGFP fusion was constructed to follow its localization in fungal cells and during infection.

Enhanced Resistance to Viruses in Nicotiana edwardsonii 'Columbia' Is Dependent on Salicylic Acid, Correlates with High Glutathione Levels, and Extends to Plant-Pathogenic Bacteria and Abiotic Stress.

Our earlier research showed that an interspecific tobacco hybrid (Nicotiana edwardsonii 'Columbia' [NEC]) displays elevated levels of salicylic acid (SA) and enhanced resistance to localized necrotic symptoms (hypersensitive response [HR]) caused by tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), as compared with another interspecific hybrid (Nicotiana edwardsonii [NE]) derived from the same parents. In the present study, we investigated whether symptomatic resistance in NEC is indeed associated with the inhibition of TMV and TNV and whether SA plays a role in this process. We demonstrated that enhanced viral resistance in NEC is manifested as both milder local necrotic (HR) symptoms and reduced levels of TMV and TNV. The presence of an adequate amount of SA contributes to the enhanced defense response of NEC to TMV and TNV, as the absence of SA resulted in seriously impaired viral resistance. Elevated levels of subcellular tripeptide glutathione (GSH) in NEC plants in response to viral infection suggest that in addition to SA, GSH may also contribute to the elevated viral resistance of NEC. Furthermore, we found that NEC displays an enhanced resistance not only to viral pathogens but also to bacterial infections and abiotic oxidative stress induced by paraquat treatments. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Induced systemic resistance against Alternaria sesami, causing blight of sesame and its toxin characterization

Different resistance inducing chemicals were tested for inhibition of growth and induction of resistance against blight in sesame. Among them, salicylic acid (10 mM) was effective in inhibiting the mycelial growth of A. sesami (68.8%) that causes the blight disease of sesame. The resistance inducing chemicals, plant extracts and bioagents tested in vivo, with challenge inoculation of A. sesami, salicylic acid at one per cent concentration was found to be effective in suppressing the pathogen and resulted in higher vigor index (1138.28) followed by P. fluorescens with good germination per cent of 97.35 and vigor index of 1029.85. The higher vigor index obtained in these treatments might be due to their support for increased germination, good root and shoot growth. Further, the pathogen produces toxic metabolite in culture, the crude toxin was extracted and analysed through infra-red analysis, the peak indicated that the compound may contain more than two OH groups and its assay on seeds and seedling ofsesame and tomato showed least germination, shoot and root length and necrotic symptoms respectively.

Early Detection of Botrytis cinerea Infection in Cut Roses Using Thermal Imaging.

Botrytis cinerea (B. cinerea) causes gray mold disease (GMD), which results in physiological disorders in plants that decrease the longevity and economic value of horticultural crops. To prevent the spread of GMD during distribution, a rapid, early detection technique is necessary. Thermal imaging has been used for GMD detection in various plants, including potted roses; however, its application to cut roses, which have a high global demand, has not been established. In this study, we investigated the utility of thermal imaging for the early detection of B. cinerea infection in cut roses by monitoring changes in petal temperature after fungal inoculation. We examined the effects of GMD on the postharvest quality and petal temperature of cut roses treated with different concentrations of fungal conidial suspensions and chemicals. B. cinerea infection decreased the flower opening, disrupted the water balance, and decreased the vase life of cut roses. Additionally, the average temperature of rose petals was higher for infected flowers than for non-inoculated flowers. One day before the appearance of necrotic symptoms (day 1 of the vase period), the petal temperature in infected flowers was significantly higher, by 1.1 °C, than that of non-inoculated flowers. The GMD-induced increase in petal temperature was associated with the mRNA levels of genes related to ethylene, reactive oxygen species, and water transport. Furthermore, the increase in temperature caused by GMD was strongly correlated with symptom severity and fungal biomass. A multiple regression analysis revealed that the disease incidence in the petals was positively related to the petal temperature one day before the appearance of necrotic symptoms. These results show that thermography is an effective technique for evaluating changes in petal temperature and a possible method for early GMD detection in the cut flower industry.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.

Exploring the Impact of Potassium on Growth, Photosynthetic Performance, and Nutritional Status of Lemon Trees (cv. Adamopoulou) Grafted onto Sour Orange and Volkamer Lemon Rootstocks

(1) Background: This study investigates the effects of potassium (K) and rootstock on the growth, photosynthetic activity, and mineral nutrition of lemon trees; (2) Methods: Lemon trees (cv. Adamopoulou) grafted onto sour orange (Citrus aurantium) (SO) or Volkamer lemon (Citrus volkameriana) (VL) rootstock were cultivated hydroponically under 0.00, 0.75, 1.50, 3.00, and 6.00 mM K. Plant growth and nutrition parameters, as well as leaf photosynthetic rate, stomatal conductance, transpiration rate, intracellular CO2, chlorophyll, and carotenoid concentration were assessed; (3) Results: Under K deficiency (0 mM K), plants exhibited chlorotic and necrotic symptoms, more pronounced in older leaves. Potassium deficiency adversely affected various physiological processes in lemon leaves, including a decrease in photosynthesis rate, stomatal conductance, transpiration rate, intercellular CO2 concentration, water use efficiency, CO2 utilization efficiency, chlorophyll a/b ratio, and carotenoid concentrations (some effects were rootstock-dependent). Low photosynthetic rates under K deficiency were due to both stomatal- and non-stomatal limitations. Elevated K in the nutrient solution consistently reduced the total plant uptake of P, Ca, Mg, B, Mn, and Zn, resulting in nutrient imbalances, as evidenced by the significant decrease in P, Ca, Mg, and Zn concentrations found in scion tissues (especially at the 6 mM K treatment). Rootstock-dependent responses were also observed in scion leaf and stem growth and in mineral nutrient concentrations, uptake, and distribution across plant parts; (4) Conclusions: Our study reveals interesting aspects on how to optimize K fertilization in lemon trees in the context of sustainable agriculture by considering nutrient interactions and rootstock-dependent effects. Understanding these complex interactions and improving K fertilization practices is expected to improve lemon tree performance, yield, and fruit quality.

First Report of Peanut Black Pod Rot Caused by Berkeleyomyces rouxiae in China.

Peanut (Arachis hypogaea L.) is an important oilseed and cash crop cultivated in over 100 countries worldwide. The major producers are China, India and USA (Ding et al. 2022). In September 2022, peanut pods exhibiting black necrotic symptoms on the shell surface were observed in Puyang, Henan Province, China. These black spots often merged to form larger necrotic spots on the shell. Disease incidence was 100% in susceptible varieties. Symptomatic shell pieces were surface sterilized with 75% ethanol for 3 min, rinsed three times with sterile water, and then transferred onto PDA medium supplemented with 25 µg/ml chloramphenicol (Long et al. 2022). Isolation frequency of a fungus with similar-appearing colonies from symptomatic pods was 81.7%. A pure culture of a representative isolate, PYHB, was obtained through single-sporing and maintained on PDA plates at 25℃ in darkness. The colony initially appeared white but turned black within 2 days. The isolate produced dark brown, unicellular chlamydospores, which were arranged in club-shaped chains consisting of two to seven cells. The size of the unicellular chlamydospores varied from 3.34 to 15.27 µm (average:6.81, n = 100) in length and 8.30 to 15.51 µm (average:11.29, n = 100) in width. The endoconidia were hyaline and cylindrical, measuring 7.91-22.94 × 1.69-4.81 µm (average: 12.16 × 3.13, n = 100). Based on morphological characteristics, the isolate was tentatively identified as a Berkeleyomyces sp. (Nel et al. 2018; Long et al. 2022). The ITS region of r-DNA, the ribosomal large subunit (LSU), the minichromosome maintenance complex component 7 (MCM7), and the 60S ribosomal protein RPL10 (60S) genes were amplified using ITS1/ITS4, LR0R/LR5, rouxMCM7-F/rouxMCM7-R and roux60s-F/roux60s-R primers, respectively (White et al. 1990; Vilgalys and Hester 1990; Nakane and Usami 2020). The sequences were deposited in GenBank (ITS: OR053803; LSU: OR053818; MCM7: OR058549; 60S: OR060656). Through BLASTn analysis of the NCBI GenBank database, the generated ITS and LSU sequences showed 100% identity to Berkeleyomyces rouxiae (GenBank MF952418.1 and MF948662.1, respectively) and B. basicola (GenBank MT221585.1 and MH868639.1, respectively). Importantly, the MCM7 and 60S sequences were 100% identical to B. rouxiae (GenBank MF967114.1 and MF967077.1, respectively). Phylogenetic analysis combining ITS, LSU, MCM7, and 60S sequences showed that the isolate PYHB clustered with B. rouxiae. To evaluate pathogenicity, surface-sterilized healthy peanut pods (n = 90) were immersed in a 1×106 spore/ml conidial suspension obtained from isolate PYHB for 5 min and placed in Petri dishes containing moistened cotton at 25°C for 10 days. Pods (n = 90) inoculated with sterile water served as controls. Inoculated pods displayed black necrosis 10 days after inoculation (dai), whereas no symptoms were observed on the control pods at 21 dai. The reisolated pathogen was shown to be identical to the original inoculum through morphological and phylogenetic analysis. Black root rot is a fungal disease caused by Berkeleyomyces spp. (syn. Thielaviopsis spp.) and affects various crops and ornamentals, such as cotton, tobacco, carrot, holly, and pansy (Rahnama et al. 2022). The causal agents B. rouxiae and B. basicola have similar morphological characteristics but can be differentiated through molecular characterization (Nel et al. 2018). To our knowledge, this is the first report of black pod rot in peanut caused by B. rouxiae in China. The finding from this study will contribute to the development of monitoring and management strategies to combat this destructive disease in peanut cultivation.

First Report of Basal Rot of Onion Caused by Fusarium oxysporum f. sp. cepae in Saudi Arabia.

Onion (Allium cepa L.) is a globally important crop worldwide including Saudi Arabia. In November 2020, 2-month-old onion plants (cv. Redwing) in commercial fields within the Sajir area of Riyadh region (∼ 1.4 ha), showed symptoms of yellowing, wilting, stunting, bulb discoloration, rot in the basal parts of bulb and decrease in roots. In the advanced stages, the affected plants collapsed and died. The incidence of symptomatic plants ranged from 30 to 65% in the surveyed fields. To isolate the pathogen, symptomatic onion plants (n =20) were sampled. Diseased tissues from roots and bulbs were cut into small pieces (4 × 4 mm), sanitized with 1% sodium hypochlorite solution for 2 min, submerged in 70% alcohol for 20 s, then rinsed with sterile water, before plating on potato dextrose agar (PDA) medium. The plates were incubated at 25°C for 6 days. Subcultures of the mycelia grown out of the diseased tissues produced purplish pink fungal colonies on PDA. On carnation leaf agar, cultures were characteristic of Fusarium oxysporum as described by Leslie and Summerell (2006), with the presence of unicellular microconidia (3.8 to 7.8 × 1.7 to 2.5 μm, n= 50) without septa in false heads or short monophialides and slightly curved macroconidia (16.3 to 28 × 4.2 to 6.1 μm, n= 50) with two to four septa. Older mycelia developed many chlamydospores that were single or in short chains. To further confirm the pathogen identification, DNA was extracted from single-spore cultures of three representative isolates using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (RPB2) DNA were amplified by PCR and sequenced with the following primers: ITS4 and ITS5 (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 2008); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and RPB2 sequences using MEGA7 placed these isolates in the F. oxysporum clade. The ITS, TEF1-α, and RPB2 sequences of an isolate FOC-OR9 were submitted to GenBank (OL721757, OL764494, and OL764495 respectively). To confirm the forma specialis cepae, a fragment of the F. oxysporum f. sp. cepae gene Secreted In Xylem 3 (SIX3) was amplified by PCR (Kalman et al. 2020). The SIX3 amplicon (∼ 277-bp) was sent for sequencing, and the sequence was submitted to GenBank (OL828265). BLASTn analysis of the sequences showed 100% identity with F. oxysporum f. sp. cepae (KP746408). To fulfill Koch's postulates, pathogenicity tests were performed with healthy onion bulbs cv. "Redwing" of 100-150 g each. Prior to inoculation of onion bulbs, the dry bulb scales, one of the fleshy inner scales, as well as the roots were removed. Bulbs were then surface sterilized (as described above) and injected with 20 µl of a conidial suspension (106 spores/ ml) into the basal plate of each bulb and approximately 1 cm deep into the tissue. Six bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 28 °C in the dark. Six bulbs injected with sterile water and six non-inoculated bulbs served as controls. At the 4th week post inoculation, necrotic rot symptoms and brown discoloration were observed on the basal plates of these inoculated bulbs (similar symptoms to those observed in the field), while control treatments showed no symptoms. The pathogen was re-isolated from the basal plates onto PDA and identified morphologically and molecularly as F. oxysporum f. sp. cepae, thus fulfilling Koch's postulates. The test was repeated twice. This pathogen was previously reported causing onion basal rot in United Kingdom (Taylor, et al., 2013). To our knowledge, this is the first report of basal rot in onion caused by F. oxysporum f. sp. cepae in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses for onion growers in Saudi Arabia. Also, Fusarium mycotoxin contamination of onion bulb could pose a public health risk.

Isolation and Identification Antagonistic Bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli.

In this study, we aimed to screen antagonistic microorganisms against Acidovorax citrulli, the causal agent of bacterial fruit blotch, which is known to induce sever diseases in cucurbit crops. From 240 bacterial strains isolated, only one unknown bacterial isolate, named YM002, showed significant antagonistic activity against A. citrulli KACC17909. Further experiments revealed that YM002 shows antagonistic activity against all tested A. citrulli strains, including KACC17000, KACC17001 and KACC17005, to different degrees. The phylogenetic analysis of 16S rRNA sequences identified YM002 as Paenibacillus tianmuensis. Importantly, pretreatment of cucumber (Cucumis sativus) leaves with YM002 enhanced disease resistance as observed by significantly reduced necrotic symptom development and bacterial growth. YM002-induced resistance accompanied by enhanced expression of defense-related genes, such as PAL1, PR1-1a and CTR1. Importantly, culture filtrate of YM002 significantly suppressed biofilm formation and swimming motility of A. citrulli, which is indispensable for its full virulence. In addition to its antagonistic activity, YM002 showed a various plant growth promotion (PGP)-related traits, such as production of ammonia production, amylase production, ACC deaminase production, inodole-3-acetic acid production, extracellular protease production, siderophore production, and zinc solubilization activities. Indeed, treatment of cucumber roots with YM002 significantly enhanced plant growth parameters, such as fresh and dry weight of leaves or roots. This study suggests the potential of YM002 as an effective PGPR with biological control activity against Acidovorax citrulli in cucumber plants.

Heat shock-induced enhanced susceptibility of barley to Bipolaris sorokiniana is associated with elevated ROS production and plant defense-related gene expression.

● Heat stress alters plant defense responses to pathogens. A short-term heat shock (HS) promotes infections by biotrophic pathogens. However, little is known about how HS affects infection by hemibiotrophic pathogens like Bipolaris sorokiniana (teleomorph: Cochliobolus sativus) (BS). We assessed the effect of HS in BS-susceptible barley (Hordeum vulgare cv. Ingrid) by monitoring leaf spot symptoms, BS biomass, reactive oxygen species (ROS) and plant defense-related gene expression following pre-exposure to HS. ● For HS, barley plants were kept at 49 °C for 20 seconds. BS biomass was assessed by qPCR, ROS levels determined by histochemical staining, while gene expression assayed by RT-qPCR. ● HS suppressed defense responses of barley to BS, resulting in more severe necrotic symptoms and increased fungal biomass, as compared to untreated plants. HS-induced increased susceptibility was accompanied by significant increases in ROS (superoxide, hydrogen peroxide). Transient expression of plant defense-related antioxidant genes and a barley programmed cell death inhibitor (HvBI-1) was induced in response to HS. However, HS followed by BS infection caused further transient increases in expression of HvSOD and HvBI-1 correlating with enhanced susceptibility. Expression of the HvPR-1b gene encoding pathogenesis-related (PR) protein-1b increased several fold at 24 hours after BS infection, however, HS further increased transcript levels along with enhanced susceptibility. ● HS induces enhanced susceptibility of barley to BS, associated with elevated ROS levels and expression of plant defense-related genes encoding antioxidants, a cell death inhibitor and PR-1b. Our results may contribute to elucidating the influence of HS on barley defense responses to hemibiotrophic pathogens.

First Report of Bacterial Necrosis Caused by Pantoea vagans in Mango in China.

Mango (Mangifera indica L.) is one of the most important tropical fruits in China. Bacterial black spot is one of the primary factors limiting mango production and thus leads to huge economic losses (Bie et al. 2022). In June 2020, necrotic symptoms similar to bacterial black spot was observed with incidence 30% to 65% on mango cultivar Yuwen, Jinhuang, Tainong and Guifei in Baise, Guangxi, China. Typically, the lesions began as chlorotic spots that coalesced into an irregular shape, becoming black and slightly raised, with a yellow halo. Thirteen diseased samples collected from five orchards were cut into approximately 5-mm pieces, sterilized for 10 s with 75% ethanol, soaked with 2% NaClO for 1 min, and rinsed in sterilized water three times. The samples were then homogenized and a 10-fold serial dilution was made before plating onto Lysogeny broth (LB) agar. After incubation at 28°C for 3 days, one representative colony that was beige to yellow in color, round, convex and smooth with entire margins from each orchard was selected for further study. Genomic DNA was extracted to amplify the 16S rRNA gene (Lane et al. 1991). The resulting 16S rRNA sequences were compared in GenBank using BLASTn and shared at least 99% identity with Pantoea spp.. Furthermore, six housekeeping genes fusA, gyrB, leuS, pyrG, rplB and rpoB partial sequences of five isolates were amplified and sequenced (Delétoile et al. 2009). The sequences were deposited in GenBank (16S rRNA: OL413424 to OL413246, OP225727-OP225728; leuS: OL441796, OL441798 to OL441801; fusA, gyrB, leuS, pyrG, rplB and rpoB: OP272638-OP272662). The five bacterial isolates were classified as P. vagans based on the phylogenetic tree of the concatenated sequences and sequences derived from different Pantoea reference isolates inferred by maximum-likelihood using MegaX software (Kumar et al. 2018). Biochemical tests showed the isolates were Gram-negative, oxidase negative, and hydrogen oxidase positive, and could use D-glucose, D-fructose, L-rhamnose, D-galactose and D-mannitose as a carbon resource (Bradbury, 1986). Pathogenicity tests were performed on mango cv. Yuwen. The representative isolate was inoculated by infiltration with sterile needleless syringes on healthy leaves and spraying onto slightly scratched leaves with bacterial suspensions (OD600=0.1) respectively (Kutschera, et al. 2019). A Xanthomonas citri pv. mangiferaeindicae (Xcm) suspension and sterilized water were used as positive and negative controls, respectively. Inoculated plants were kept with 90 ± 5% relative humidity and 28 ± 1°C in the greenhouse for 1 week. Black to brown necrotic symptoms were observed on all leaves inoculated by infiltration except the negative control. These were observed in plants inoculated by spraying only after 2 weeks. Bacteria re-isolated from diseased tissues were consistent with the inoculated isolates and identified as P. vagans, fulfilling Koch's postulates. To date, P. vagans have been isolated from eucalyptus with bacterial blight and dieback, and maize with brown stalk rot (Brady et al. 2009). However, to our knowledge, this is the first report of P. vagans causing bacterial necrosis on mango in China. It was also found that some of the diseased samples were coinfected with P. vagans and Xcm in our investigation. Therefore, it is necessary to further study the infection mechanisms of this pathogen.