First report of Trichoderma afroharzianum causing seed rot on maize in Italy

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 60,602,320 t, covering 588,597 ha (ISTAT 2021). Trichoderma species are widespread filamentous fungi in soil, well known and studied as biological control agents (Vinale et al., 2008). Seeds of a yellow grain hybrid (class FAO 700, 132 days) were collected in September 2020 from an experimental field located in Carmagnola (TO, Italy: GPS: 44°53'11.0"N 7°40'60.0"E) and tested with blotter test (Warham et al., 1996) to assess their phytosanitary condition. Over the 400 seeds tested, more than 50% showed rotting and development of green mycelium typical of the genus Trichoderma. Due to the high and unexpected percentage of decaying kernels, ten colonies were identified by morphological and molecular methods. Single conidia colonies of one Trichoderma (T5.1) strain were cultured on Potato Dextrose Agar (PDA) for pathogenicity tests, and on PDA and Synthetic Nutrient-Poor Agar (SNA) for morphological and molecular identification. The colonies grown on PDA and SNA showed green, abundant, cottony, and radiating aerial mycelium, and yellow pigmentation on the reverse. Colony radius after 72 h at 30°C was of 60-65 mm on PDA and of 50-55 mm on SNA. The isolates produced one cell conidia 2.8 - 3.8 µm long and 2.1 - 3.6 µm wide (n=50) on SNA. Conidiophores and phialides were lageniform to ampulliform and measured 4.5 – 9.7 µm long and 1.6 – 3.6 µm wide (n=50); the base measure 1.5 – 2.9 µm wide and the supporting cell 1.4 – 2.8 µm wide (n=50). The identity of one single-conidia strain was confirmed by sequence comparison of the internal transcribed spacer (ITS), the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (Oskiera et al., 2015). BLASTn searches of GenBank using ITS (OL691534) the partial tef-1α (OL743117) and rpb2 (OL743116) sequences of the representative isolate T5.1, revealed 100% identity for rpb2 to T. afroharzianum TRS835 (KP009149) and 100% identity for tef-1α to T. afroharzianum Z19 (KR911897). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile H2O to 1×106 CFU/ml. Twenty-five seeds were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 23°C under a seasonal day/night light cycle. Twenty primary ears were inoculated, by injection into the silk channel, with 1 ml of a conidial suspension of strain T5.1 seven days after silk channel emergence (BBCH 65) (Pfordt et al., 2020). Ears were removed four weeks after inoculation and disease severity, reaching up to 75% of the kernels of the twenty cobs, was assessed visually according to the EPPO guidelines (EPPO, 2015). Five control cobs, inoculated with 1 ml of sterile distilled water were healthy. T. afroharzianum was reisolated from kernels showing a green mold developing on their surface and identified by resequencing of tef-1α gene. T. afroharzianum has been already reported on maize in Germany and France as causal agent of ear rot of maize (Pfordt et al. 2020). Although several species of Trichoderma are known to be beneficial microorganisms, our results support other findings that report Trichoderma spp. causing ear rot on maize in tropical and subtropical areas of the world (Munkvold and White, 2016). The potential production of mycotoxins and the losses that can be caused by the pathogen during post-harvest need to be explored. To our knowledge this is the first report of T. afroharzianum as a pathogen of maize in Italy.

Neofusicoccum mediterraneum Is Involved in a Twig and Branch Dieback of Olive Trees Observed in Salento (Apulia, Italy)

Olive trees are infected and damaged by Botryosphaeriaceae fungi in various countries. The botryosphaeriaceous fungus Neofusicoccum mediterraneum is highly aggressive and is a major concern for olive groves in Spain and California (USA), where it causes ‘branch and twig dieback’ characterized by wood discoloration, bark canker, and canopy blight. During surveys of olive groves in Apulia (southern Italy), we noticed that—in some areas—trees were heavily affected by severe branch and twig dieback. In addition, chlorosis and the appearance of red-bronze patches on the leaf preceded the wilting of the foliage, with necrotic leaves persisting on the twigs. Given the severity of the manifestation in zones also subject to olive quick decline syndrome (OQDS) caused by Xylella fastidiosa subsp. pauca, we investigated the etiology and provide indications for differentiating the symptoms from OQDS. Isolation from diseased wood samples revealed a mycete, which was morphologically and molecularly identified as N. mediterraneum. The pathogenicity tests clearly showed that this fungus is able to cause the natural symptoms. Therefore, also considering the low number of tested samples, N. mediterraneum is a potential causal agent of the observed disease. Specifically, inoculation of the twigs caused complete wilting in two to three weeks, while inoculation at the base of the stem caused severe girdling wedge-shaped cankers. The growth rate of the fungus in in vitro tests was progressively higher from 10 to 30 °C, failing to grow at higher temperatures, but keeping its viability even after prolonged exposure at 50 °C. The capacity of the isolate to produce catenulate chlamydospores, which is novel for the species, highlights the possibility of a new morphological strain within N. mediterraneum. Further investigations are ongoing to verify whether additional fungal species are involved in this symptomatology.

Characterization and fungicides sensitivity of Phytophthora cinnamomi isolates causing avocado root rot in Zitácuaro, Michoacán

<em>Phytophthora cinnamomi</em> is the pathogen most frequently associated with avocado root rot. In Zitácuaro, Michoacán, production has increased by 19.8%; however, there are no studies of root rot in this area. The objective of the study was to characterize the isolates obtained from avocado roots and assess the sensitivity to fungicides. Samples from 5 avocado orchards were collected, sampling 5 trees per orchard (a total of 25 samples). The samples isolated were characterized morphological and molecularly. Mating type was analyzed using reference isolates of<em> P. cinnamomi</em> A1 (isolate from camelia) and A2 (isolate from avocado). To confirm the pathogenicity, tests were performed on avocado fruits with the isolates. The sensitivity of 15 isolates to potassium phosphite and to metalaxyl-M at different concentrations was evaluated<em> in vitro</em>. In a subgroup of six isolates, it was evaluated whether there was a relationship between growth rate and potassium phosphite sensitivity. Fifteen isolates were obtained with coenocytic coraloid mycelium, chlamydospores, sporangia without papilla, ovoid to ellipsoid, with internal proliferation, heterothallic with mating type A2, with amphigynous antheridia and plerotic oospores, characteristics consistent with <em>P. cinnamomi</em>. The inoculated isolates were pathogenic on avocado fruits. The isolates were more sensitive to potassium phosphite than to metalaxyl-M, with mean EC50 values of 24.62 and 0.215 ?g mL-1 of i.a., respectively. No relationship was observed between growth rate and potassium phosphite sensitivity. It is necessary to obtain a greater number of<em> P. cinnamomi</em> isolates for virulence studies.

Aqueous extracts from Acalypha gaumeri and Bonellia flammea for leaf blight control in chrysanthemum (Chrysanthemum morifolium)

The chrysanthemum is the second most important cut flower in the world, however, its quality and commercial value is affected by the leaf blight produced by <em>Alternaria </em>spp. The objective of this work was to evaluate the causal agent of leaf blight in Chrysanthemum, and its control with aqueous extracts of <em>Acalypha gaumeri </em>and <em>Bonellia flammea</em>. The fungus was collected and identified from leaves and stems of chrysanthemum plants. Subsequently, molecular identification and pathogenicity tests were performed on chrysanthemum plants. In the field, treatments were evaluated with weekly applications of: T1: <em>B. flammea </em>bark extract, T2: <em>A. gaumeri </em>root extract, T3: negative control (water) and T4: Captan® fungicide. Prior to the application of the treatments, plants were inoculated with the isolated fungus (2.5 × 106 spores mL-1) and severity was evaluated. <em>Alternaria chrysanthemi </em>was identified as the causal agent. Based on the severity percentage, the lowest averages of the area under the disease progress curve, the lowest rates of apparent infection, the lowest intensity of the disease and the greater effectiveness in controlling the disease were observed for T2 (165, 0.017, 8 and 67%, respectively) followed by T1 (186, 0.022, 13 y 50 %, respectively) and T4 (179, 0.023, 14 y 45%, respectively), observing a significantly different than negative control T3 (369, 0.025, 25 and 0%, respectively). Plant extracts have potential to be used as an alternative in the management of <em>Alternaria </em>leaf blight in chrysanthemum.

Pathogenicity of Phytophthora × alni Isolates Obtained from Symptomatic Trees, Soil and Water against Alder

Phytophthora alni complex (P. × alni, P. × multiformis, P. uniformis) are pathogens attacking alder seedlings and trees, causing significant losses in nurseries and natural tree stands. Decay of alder trees has been observed in Poland for over a dozen years. Overall, 25 Polish isolates of P. × alni obtained from symptomatic alder trunks, rhizosphere soil surrounding infected trees, and nearby natural streams were compared with isolates from symptomatic trunks obtained in France, Belgium and Hungary. Morphologic characterization of mycelium, vegetative and generative organs, temperature effect on mycelium growth, and their pathogenicity were studied. The mycelium growth rate of isolates from symptomatic plants was fastest on Carrot Agar (CA) medium, and from soil and water on Vegetable Agar (V8A) medium. The sizes of zoosporangia varied depending on their origin. The isolates that originated from the soil had the largest zoosporangia. The diameter of the oogonia and antheridia did not differ regardless of their origin. The results of pathogenicity tests of P. × alni isolates obtained from different sources showed that the soil isolates were the most aggressive in each test, followed by the isolates from the trunks and water. A simple test of leaf colonization can give an idea of the aggressiveness of the isolate towards the shoots and roots. No morphological or physiological markers of aggressiveness have been found.

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens.

Molecular Characterization, Pathogenicity and Biological Characterization of Colletotrichum Species Associated with Anthracnose of Camellia yuhsienensis Hu in China

Camellia yuhsienensis Hu, a species of tea oil tree with resistance to anthracnose, is widely used to breed disease-resistant Camellia varieties. In 2019, anthracnose symptoms were observed on Ca. yuhsienensis for the first time. However, the species and biological characteristics of Colletotrichum spp. isolated from Ca. yuhsienensis (YX-Colletotrichum spp.) have not been elucidated. In this study, five isolates (YX2-5-2, 2YX-3-1, 2YX-5-1, 2YX-8-1-1 and 2YX-8-1-2), which were consistent with the morphological characteristics of Colletotrichum spp., were obtained from Ca. yuhsienensis. A phylogenetic analysis demonstrated that YX2-5-2, 2YX-3-1 and 2YX-8-1-2 belonged to first clade along with Colletotrichum fructicola. 2YX-8-1-1 belonged to the second clade along with Colletotrichum siamense. 2YX-5-1 belonged to the third clade with Colletotrichum camelliae. Pathogenicity tests revealed that the pathogenicity of YX-Colletotrichum spp. was stronger than that of Colletotrichum spp. isolated from Camellia oleifera (GD-Colletotrichum spp.). Biological characteristics illustrated that the mycelial growth of YX-Co. camelliae (2YX-5-1) was slower than that of GD-Co. camelliae when the temperature exceeded 20 °C. In addition, in the presence of ions, the mycelial growth of YX-Co. fructicola (YX2-5-2) and YX-Co. siamense (2YX-8-1-1) was also slower than that of GD-Co. fructicola and GD-Co. siamense. Furthermore, the ability of YX-Colletotrichum spp. to utilize lactose and mannitol was weaker than that of GD-Colletotrichum spp., while the ability to utilize NH4+ was generally stronger than that of GD-Colletotrichum spp. This is the first report of anthracnose of Ca. yuhsienensis induced by Co. fructicola, Co. siamense and Co. camelliae in China. These results will provide theoretical guidance for the study of the pathogenesis and control of anthracnose on Ca. yuhsienensis.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Cockscomb (Celosia argentea var. cristata) in Taiwan

Cockscomb (Celosia argentea) is commonly found in subtropical and temperate zones of Africa, South America and South East Asia, and is a popular ornamental plant in the family Amaranthaceae. Cockscomb has been known to contain antiviral proteins, betalains, and anthocyanin, which can be applied in beneficial ways (2). In September 2020, a cockscomb plant (Celosia argentea var. cristata) showing typical galling root symptoms likely infected by root-knot nematodes (Meloidogyne sp.) was collected from a garden in Taichung, Taiwan, and a quick exam of several individuals using MK7F/R primers (7) indicating they were M. enterolobii. Nematode population was established from a single egg mass and was later used for species identification and pathogenicity tests. Five perineal patterns of mature females from the single female population show round to oval shapes with weak lateral lines. Dorsal arches are moderate to high, almost squared, with the smooth ventral striae. Second-stage juveniles are vermiform and have a slender tail, tapering to rounded tip with distinct hyaline region at the tail terminus. Morphological measurements of 28 J2s revealed body length = 457.2 ± 20.6 (416.1-506.9) μm, body width = 16.0 ± 2.0 (13.4-20.3) μm, stylet length = 14.7 ± 0.5 (13.9-15.9) μm, dorsal gland orifice to the stylet base = 4.0 ± 0.5 (2.0-4.8) μm, and tail length = 56.0 ± 3.8 (47.4-60.3) μm. Female perineal patterns and morphometric data are similar to the original description of Meloidogyne enterolobii (9). DNA purified from approximately 1500 juveniles using GeneMark Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan) was used to amplify 18S rDNA fragment, D2-D3 expansion segments of 28S rDNA, and a COII region on mtDNA with primer sets 1A/MelR, D2A/D3B, and C2F3/1108, respectively (4,5,6). The 18S rDNA sequence (OK076893) of this study shares 99.94% nucleotide identity with those of M. enterolobii isolated from the United States (KP901058) and China (MN832688). D2D3 sequence of haplotype 1 (OK076898) shows 100% identity to those of M. enterolobii from China (MT193450) and Taiwan (KP411230). Sequence of haplotype 2 (OK076899) shows 99.86% identity to those of M. enterolobii from the United States (MN809527) and China (MN269945). Sequence of the COII region (OK086042) show 99.86% identity to that of M. enterolobii from China (MN269945). Phylogenetic trees of the three gene sequences were plotted following Ye et al.(10), revealing that the newly described root-knot nematode on Cockscomb is grouped with other M. enterolobii isolates. DNA fragment amplified by primer sets Me-F/R(3) and MK7F/R specifically targeting of M. enterolobii yielded 236 bp and 520 bp, respectively. Pathogenicity tests were assayed, from July to September 2021, on three-week-old nematode-free cockscomb plant directly germinated from seeds of SkyStar® (ASUSA SPIKE SEEDS, Taipei, Taiwan) planted in a 10.5 cm diameter pot filled with 600 ml sterilized peat moss: sand (1:1, v/v) soil in a 28℃walk-in chamber. Nematode eggs were extracted using 0.05% NaoCl as described by Vrain(8), and cockscomb plants (n=3) were inoculated by adding 6000 eggs (10 eggs/ cm3). Cockscomb plants treated with water were used as mock controls. Rf value of the inoculated plants were determined by the method of Belair and Benoit (1) 45 days after inoculation, and the average was 4.13. No galls were observed on the roots of control plants. The results confirmed that cockscomb is the new host of M. enterolobii. To the best of our knowledge, this is the first report of M. enterolobii on Celosia argentea var. cristata in Taiwan.

Characterization of Xanthomonas citri pv. glycines Population Genetics and Virulence in a National Survey of Bacterial Pustule Disease in Korea

Xanthomonas citri pv. glycines (Xcg) is a major pathogen of soybean (Glycine max) in South Korea, despite the availability of soybean varieties with some resistance. We conducted a nationwide survey of the incidence and severity of bacterial pustule caused by Xcg. The percentage of infected fields was 7% to 17% between 2015 and 2017. We characterized the diversity of a nationwide collection of 106 Xcg isolates based on avrBs3 banding patterns. The isolates fell into 11 groups, each represented by a type strain; only two of these were similar to isolates collected from 1999 to 2002. The diversity of Xcg strains increased and the dominant strains changed between 1999 and 2017, with three new type strains comprising 44% of the isolates examined in 2012 to 2017. Pathogenicity tests did not show evidence for a shift in the races or aggressiveness of Xcg strains. Korean soybean cultivars, including the widely-grown Daewon cultivar, were susceptible to the 11 new type strains. The cultivar CNS, which carries the rxp resistance gene, was susceptible to most type strains, including two representing 83% of the Korean Xcg strains. In contrast, Williams 82, which also carries rxp, showed resistance to at least five type strains. Collectively, these results suggest that Williams 82 has resistance loci in addition to rxp. The widespread distribution of Xcg, the high virulence of the current endemic strains, and the low resistance of most Korean soybean cultivars collectively favor widespread disease in Korea in years that are favorable to pustule development.

Virulence and indole-3-acetic acid (IAA) biosynthesis ability of Turkish Pseudomonas savastanoi pv. savastanoi isolates and susceptibility of some native olive genotypes

Aim of study: To evaluate the virulence and indole-3-acetic acid (IAA) biosynthesis ability of several Turkish P. savastanoi pv. savastanoi isolates and the susceptibility of some native genotypes to olive knot. Area of study: The Aegean, Marmara, and Mediterranean Regions of Turkey. Material and methods: 101 isolated bacteria were identified on the basis of biochemical, PCR for amplification of the bacterial iaaL gene, and pathogenicity tests. The virulence of the isolates was determined in a randomized experimental trial carried out by stem inoculation of pot-grown seedlings of olive (cv. ‘Manzanilla’) in the growing chamber. The amounts of IAA produced by the isolates were determined colorimetrically. The susceptibility of native olive genotypes was evaluated on 2-yr old plants inoculated with two distinct strains. Main results: Tested P. savastanoi pv. savastanoi isolates showed significant differences in virulence found to be associated with their geographical origin. The isolates produced IAA amounts varied from 148.67 to 0.3 μg mL-1. The geographical variation in IAA biosynthesis ability of the isolates was observed. No correlation (R=0.0225) was determined between virulence and IAA amounts of the isolates. Native olive genotypes indicated different susceptibility levels to the olive knot pathogen. No genotype tested had complete resistance. However, low susceptible genotypes (‘Memecik’, ‘Ayvalık’ and ‘Uslu’) were identified. Some genotypes had variable reactions depending on the isolate used. Research highlights: The results undergird the differences in the virulence and IAA production of the isolates within the area and also between geographical locations. Genotypes with low susceptibility can be used as genitors in further breeding studies.