Morpho-Molecular Identification, Virulence and In Vitro Sporulation of Fungi Associated with Maize Foliar Diseases in Egypt

Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software

Circular or irregularly shaped necrotic spots with yellow border, present on ca. 30% of the leaves were observed in 2010 on pomegranate trees (Punica granatum) cv. Wonder- ful in an orchard in Thessaloniki (northern Greece). Single- spore cultures from the fungus isolated on PDA gave rise to white colonies which turned to grayish-black and produced obclavate, obpyriform or ellipsoidal conidia in long chains. Conidia had 1 to 6 transverse and 0 to 3 longitudinal septa and measured 9.4-30.8×5.6-15.4 μm (average 20.1×10.7 μm). These morphological characteristics conform to those of Alternaria alternata (Fr.) Keissl. (Simmons, 2007). The ITS1- 5.8S-ITS2 region was amplified with primers ITS1 and ITS4 and sequenced (GenBank accession Nos KT900855- KT900859). A BLAST search of GenBank database revealed 100% homology with the sequences of various A. alternata isolates (e.g. KR149266). Spraying leaves of 10 healthy green- house-grown pomegranate plants (cv. Wonderful) with a 106 spores/ml spore suspension resulted in the production, 20 days post inoculation, of leaf spots similar to those observed in the field. Control plants sprayed with sterile distilled wa- ter remained symptomless. A. alternata was reisolated from artificially inoculated leaves fulfilling Koch’s postulates. Al- though A. alternata has been reported to cause fruit rot on pomegranate in Greece (Tziros et al., 2008) this is the first report of A. alternata causing a foliar disease on this host.

During the Summer 2013, a new foliar disease was observed on taro (Colocasia esculenta) in tropical areas of Iran, including Hormozgan, where many farmers were alarmed by complete destruction of their taro cultivation. Leaf spots, yellow to dark brown, were subglobose and oval. A fungus was consistently isolated from diseased tissues on Potato Dextrose Agar (PDA) at 23± 2°C. Morphological observations were carried out on Synthetic Nutrient Agar (SNA) medium as described by Bensch et al. (2012). Conidiophores were macronematous, cylindrical-oblong with several nodes being quite apart from each other, very long, up to 350 μm or even longer. Conidia ellipsoid-subcylindrical to cylindrical, forming short, unbranched or branched chains. Terminal conidia were 9–16 × 5–8 μm and aseptate or 1-septate. A portion of the translation elongation factor 1-α gene was amplified (Bensch et al., 2012) and sequenced (GenBank Accession No. KP893386). A BLAST search showed 99% similarity with Cladosporium colocasiae sequences deposited in GenBank. To verify Koch’s postulates, pathogenicity was tested by spraying a conidial suspension prepared from cultures on PDA (1 x106 spores ml ) in sterile water on healthy leaves of four test plants (5- 7 leaf stage, cv. Niue). Control plants were sprayed with sterile water. Plants were kept in a greenhouse (average minimum temp. 24°C, max. 37°C and relative humidity 64%). Symptoms appeared 2 weeks post-inoculation on inoculated plants, whereas control plants remained asymptomatic. Reisolation of the pathogen with the same morphological characters was achieved from symptomatic plants only, hence confirming the causal agent as C. colocasiae. To our knowledge, this is the first report of C. colocasiae on C. esculenta in Iran.

Hevea brasiliensis (natural rubber) is one of the major economically important plantation crops in Sri Lanka. Traditionally, rubber was grown mainly in the wet zone of the country. Currently, the cultivation is being expanded to the intermediate and dry zone. These new rubber growing areas are known as non-traditional rubber growing areas. Corynespora leaf fall disease is regarded as the most destructive foliar disease and the disease has caused a major devastation in rubber industry resulting in a remarkable economic lass of Sri Lankan rubber industry. The causative agent is the fungus, Corynespora cassiicola. Characteristic symptom of the fungus on rubber leaves is the “railway track lesion”. Later more than ten different symptoms have been reported for this disease. The symptom varied based on the type of clone, maturity level and also on the environmental condition. Due to this reason, the disease identification has become complicated especially among the field staff. This study was carried out to report newly produced symptoms of this disease. Twelve different symptoms have been illustrated with the most characteristic symptom. In this study to collect Corynespora leaf disease samples from non-traditional rubber growing areas among the lesion and unusual symptoms was observed from Padiyathalawa. Then the fungus was isolated on to PDA. Later single spore isolation technique was employed to purify the culture. Koch’s postulates were proven. The fungus was identified as based on cultural and reproductive characteristics. The newly reported symptom is illustrated to aid disease diagnosis. Keywords: Hevea brasiliensis, Non-traditional areas, Symptoms, Corynespora cassiicola, Foliar disease

The jackfruit is also known as jack tree, or sometimes simply jack is a species of tree in the mulberry family. It has pointed projections outside and soft flesh inside, which is intensely sweet and delicious in taste. The causal organism is a green parasitic alga whose usual hosts are plants with leathery leaves such as cotoneasters, magnolias, hollies, rhododendrons, and viburnums. Algal leaf spot is a foliar disease most commonly seen in warm, humid climates or in greenhouses. Discoloration and darkening of the bark from the tip downwards are the main symptoms of the disease. The spot on the leaf is irregular in shape and size, light to dark brown surrounded by diffused chlorotic yellow hallow, marginal leaf tissues become black, necrotic, and gradually spread to the leaf center. The pathogen has septate mycelia with inter and intracellular haustoria. Sexual spores are the ascospores borne in ascus, and the asexual spores are Conidia borne in sporangia.

On 29 October 2018, a leaf spot disease was observed on strawberry leaves (Fragaria × ananassa Duch., cultivar Portola) on organically grown plants in high plastic tunnels in Santa Maria, California. Symptoms consisted of circular lesions measuring 2 to 3 cm in diameter, concentric rings with a darker brown outer ring and fungal sporulation in the center of the lesion. The diseased leaf sample was surface sterilized in 10% sodium hypochlorite for 1 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) after drying. The resulting culture produced conidia that were hyaline and round to ellipsoid (8 to 14 × 5 to 8 µm, n = 50) and sclerotia that were hard, dark, round to oblong, and measured 2 to 12 × 2 to 9 mm (n = 50). Based on its morphological characteristics in culture, the isolate was putatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). The isolate produced an average of 133 sclerotia per Petri dish (100 × 15 mm; n = 3) with PDA, an uncommon feature of B. cinerea isolates from strawberry. Koch’s postulates were performed using two inoculation methods: (i) Botrytis agar plug on leaf; and (ii) conidial suspension on leaf. Leaf inoculations used a spore suspension containing 1 × 10⁶ conidia/ml and were replicated three times. Leaf inoculations using an agar plug or spore suspension were allowed to dry, misted with water, and covered with a plastic bag to maintain continuous moisture. Agar-plug inoculated leaves produced symptoms in 4 days, whereas the spore-suspension inoculations produced symptoms in 9 days. Leaf symptoms matched those observed in the field. The pathogen was reisolated from symptomatic tissue, and its morphological features matched those of the original culture. In both inoculation methods, the noninoculated controls did not result in any disease symptoms. Cross pathogenicity to leaves using an isolate of B. cinerea collected from fruit was successful in causing foliar disease, but symptoms lacked concentric rings. To determine the fungal species, total genomic DNA of five subcultures of the original field isolate was extracted using a DNeasy Plant Mini Kit (Qiagen). The heat shock protein (HSP60) (Hsp60for+/Hsp60 rev+ primers), RNA polymerase II-binding (RPB2) (RPB2for+/RPB2 rev+ primers), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (G3PDHfor+/G3PDH rev+ primers) genes were amplified (Staats et al. 2004). The PCR amplicons were purified and sequenced. The sequences were processed using BLAST in the National Center for Biotechnology, and results showed 100% homology with the HSP60 (MH796663), RPB2 (MH479932), and G3PDH (MH479930) sequence for isolates of B. cinerea. The sequences were deposited in GenBank with accession numbers MK919494 (HSP60), MK919495 (RPB2), and MK919496 (G3PDH). B. cinerea is a common pathogen of strawberry fruit and can cause gray mold, postharvest rot, and dry crown rot but has not been reported as causing a leaf spot. This disease is rare and considered of minor significance to strawberry production. However, it could be of greater significance in protected culture where humidity is higher. To our knowledge, this is the first report of B. cinerea causing strawberry leaf spot in California or elsewhere.

A foliar disease simulation model to assist the design of new control methods against black leaf streak disease of banana

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is a damaging foliar fungal disease in many soybean-growing areas of the world. Strategies to manage SBR include the use of foliar fungicides. Fungicide types, the rate of product application, and the number and timing of applications are critical components for successful rust management. The objectives of this study were to determine i) the sensitivity of P. pachyrhizi isolates collected in the U.S. to a range of fungicides and ii) the reduction of fungal infection based on fungicide type and timing of applications on soybean. There were differences (P < 0.05) in effective concentration (EC50) values among the fungicides tested. Azoxystrobin had low EC50 values for both urediniospore germination and fungal sporulation on inoculated leaflets. There were differences (P < 0.05) in fungal sporulation for application times, fungicide treatments, and their interaction when the fungus was inoculated on plants. All application times and nearly all fungicide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Information on fungicide sensitivity of P. pachyrhizi isolates and the preventive and curative effects of different fungicides are important in the management of SBR.

A pinta-preta é a principal doença fúngica foliar do mamoeiro, sendo o controle químico necessário para se conseguir produção comercial econômica. Medidas de controle alternativas aos fungicidas são necessárias para a produção sustentável de frutos de mamão de melhor qualidade. Objetivou-se, neste trabalho, determinar as condições de temperatura adequadas ao crescimento micelial e à esporulação in vitro de fungos hiperparasitas do agente causal da pintapreta do mamoeiro, potenciais agentes de biocontrole. Foram avaliados 18 isolados fúngicos (oito de Hansfordia pulvinata e dez de Acremoniumspp.), cultivados em meio BDA, às temperaturas de 15 ºC, 20 ºC, 23 ºC, 25 ºC, 27 ºC e 30 ºC, sob fotoperíodo de 12 horas. Observou-se, para isolados de H. pulvinata, que temperaturas amenas (em torno de 21 ºC) propiciam máximas de crescimento micelial e esporulação. Para os isolados de Acremonium spp., o ponto ótimo para crescimento micelial e esporulação ocorre no intervalo de 20-25 ºC.

Cercospora leaf spot caused by Cercospora beticola is one of the most damaging foliar diseases of sugar beet. The sterol demethylation inhibitor (DMI) fungicide tetraconazole is widely-used to manage Cercospora leaf spot. However, there has been an increase in prevalence of tetraconzole-resistant isolates in recent years. Knowledge about the stability of tetraconazole resistance in tetraconzole-resistant isolates after exposure to cold temperatures in the absence of the selection pressure imposed by tetraconazole application would be important information for fungicide resistance management. To explore this, we inoculated sugar beet plants with two known tetraconazole-sensitive and two known tetraconazole-resistant isolates of C. beticola . Four weeks after inoculation, symptomatic leaves were harvested and subsequently exposed to six different temperature/time treatments: -20oC (4 weeks), 4oC (4 weeks), 20oC (4 weeks), -20oC (2 weeks)/4oC (2 weeks), -20oC (1 week)/4oC (1 week)/-20oC (1 week)/4oC (1 week), and -20oC (1 week)/20oC (1 week)/-20oC (1 week)/20oC (1 week). Subsequently, spore production, spore germination, radial mycelial growth, sensitivity to tetraconazole, and disease severity were evaluated for each isolate and compared to the control (the same fungal isolates used in the inoculation series and maintained on CV8 media). After exposure to all temperature/time treatments, all tested isolates were found stable for the parameters evaluated. However, the tetraconazole-resistant isolate 09-347 after exposure to -20 oC and -20 oC /4 oC /-20 oC /4 oC treatments became 38.6 and 32.8 times more sensitive to tetraconazole , respectively. Taken together, cold temperatures do not appear to impart a significant fitness penalty in C. beticola .

Southern leaf blight (SLB) is a foliar disease caused by the fungus Cochliobolus heterostrophus infecting maize plants in humid, warm weather conditions. SLB causes production losses to corn producers in different regions of the world such as Latin America, Europe, India, and Africa. In this paper, we demonstrate a non-destructive method to quantify the signs of fungal infection in SLB-infected corn plants using a deep UV (DUV) fluorescence spectrometer, with a 248.6 nm excitation wavelength, to acquire the emission spectra of healthy and SLB-infected corn leaves. Fluorescence emission spectra of healthy and diseased leaves were used to train an Autoencoder (AE) anomaly detection algorithm-an unsupervised machine learning model-to quantify the phenotype associated with SLB-infected leaves. For all samples, the signature of corn leaves consisted of two prominent peaks around 450 nm and 325 nm. However, SLB-infected leaves showed a higher response at 325 nm compared to healthy leaves, which was correlated to the presence of C. heterostrophus based on disease severity ratings from Visual Scores (VS). Specifically, we observed a linear inverse relationship between the AE error and the VS (R2 = 0.94 and RMSE = 0.935). With improved hardware, this method may enable improved quantification of SLB infection versus visual scoring based on e.g., fungal spore concentration per unit area and spatial localization.

A foliar disease of holly (Ilex aquifolium), observed in November 2009 in a natural ecosystem in the region of Thessaloniki (northern Greece), consisted of circular or irregularly shaped necrotic spots with yellow border, 3-7 mm in diameter, present on ca. 30% of the leaves. Small pieces of tissue at the margin of the spots were excised, surface-disinfected (1% sodium hypochlorite for 1 min) and plated on potato dextrose agar. Two single-spore cultures in potato carrot agar, gave rise to initially white colonies turning grayish-black due to abundant sporulation. Conidiophores were green to brown, short, septate, branched or unbranched. Conidia, produced in single or more often branched chains, were obpyriform, with a conical or cylindrical beak, ovoid or ellipsoidal, measured 7.7-27.4x5.6-15.0 μm (average 16.3x8.8 μm), and showed 1 to 5 transverse and 0 to 3 longitudinal septa. These morphological traits and measurements tally with those of Alternaria alternata (Fr.) Keissl. (Simmons, 2007). The ITS1-5.8S-ITS2 region of the two single-spore isolates, amplified with primers ITS1 and ITS4 and sequenced (GenBank accession Nos. JQ809323, JQ809324), revealed 100% homology with the sequence of various A. alternata isolates (e.g. JQ070079). Eight 3-year-old holly plants were sprayed with a 106 conidia/ml spore suspension, covered with a polyethylene bag for 48 h and placed in a greenhouse at 23±2°C together with sterile distilled water-sprayed controls. Leaf spots like those observed in the field developed on all inoculated plants but not on controls. A. alternata was reisolated from artificially inoculated leaves. Holly infections by A. alternata have previously been reported from Poland (Orlikowski and Szkuta, 2004), the USA and Canada (Sinclair et al., 1987) but, to our knowledge, never from Greece.

Spot form of net blotch (SFNB), caused by the fungus Pyrenophora teres f. maculata, was first described in Denmark in the 1960s and is now a prevalent foliar disease of barley in many countries. This disease should be controlled as a separate disease-causing organism from the net form of net blotch (NFNB), which is caused by P. teres f. teres. The increase in prevalence of SFNB is primarily due to stubble retention and cultivation of susceptible varieties, which have resulted in increased inoculum. Infected barley stubble is the primary inoculum source for SFNB, producing both asexual spores (conidia) and sexual spores (ascospores) from pseudothecia. Spot form of net blotch causes significant losses in grain yield and quality in situations where inoculum is present, susceptible varieties are cultivated, and where the climate is cool and moist. Cultivation of resistant varieties is the most cost-effective method for control of SFNB and more than 12 different resistance sources have been identified in barley germplasm and wild barley relatives. The resistance loci of 11 of these have been mapped. Control of SFNB can also be achieved with application of foliar fungicides, crop rotation, and stubble destruction.

The fungal pathogen Cochliobolus carbonum (anamorph, Bipolaris zeicola) causes Northern Leaf Spot, leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China. Asexual spores (conidia) play a major role in both epidemics and pathogenesis of Northern Leaf Spot, but the molecular mechanism of conidiation in C. carbonum has remained elusive. Here, using a map-based cloning strategy, we cloned a single dominant gene, designated as BZcon1 (for Bipolaris zeicola conidiation), which encodes a predicted unknown protein containing 402 amino acids, with two common conserved SANT/Myb domains in N-terminal. The BZcon1 knockout mutant completely lost the capability to produce conidiophores and conidia but displayed no effect on hyphal growth and sexual reproduction. The introduced BZcon1 gene fully complemented the BZcon1 null mutation, restoring the capability for sporulation. These data suggested that the BZcon1 gene is essential for the conidiation of C. carbonum.

Tar spot caused by the fungus Phyllachora maydis Maubl. is a significant foliar disease of corn (Zea mays L.). Threatening corn production across the Americas, this disease can reduce the quality of silage and grain yield (Rocco da Silva et al. 2021; Valle-Torres et al. 2020). Lesions caused by P. maydis are usually black, glossy, and raised stromata on the leaf surface and occasionally on the husk. (Liu 1973; Rocco da Silva et al. 2021). Samples consistent with tar spot of corn were collected between September and October of 2022 from 6 fields in Kansas, 23 in Nebraska, and 6 in South Dakota. One sample was selected from each of the three states for further microscopic evaluation and molecular analysis. Signs of the fungus were visually and microscopically confirmed in eight Nebraska counties in October 2021; however, in the 2021, season tar spot sings were not found in Kansas and South Dakota. In the 2022 season disease severity varied by location; some fields in Kansas had <1% incidence, whereas in South Dakota disease incidence approached 1-2%, and in Nebraska between <1-5%. Stromata were present on both green and senescing tissues. Morphological characteristics of the pathogen were similar and consistent with the description of P. maydis (Parbery 1967) from all examined leaves across all locations. Asexual spores (conidia) were produced in pycnidial fruiting bodies ranging in size 1.29 to 2.82 x 8.84 to 16.95 µm (n = 40, average 1.98 × 13.30 μm). The pycnidial fruiting bodies were often found adjacent to perithecia within the stromata. For molecular confirmation, stromata were aseptically removed from leaves collected at each location and DNA extracted using a phenol chloroform method. The internal transcribed spacer (ITS) regions of the ribosomal RNA gene were sequenced using ITS1/ITS4 universal primers (Larena et al. 1999). Amplicons were Sanger sequenced (Genewiz, Inc., South Plainfield, NJ), and a consensus sequence for each sample was deposited in GenBank: Kansas (OQ200487), Nebraska (OQ200488), and South Dakota (OQ200489). Using the BLASTn, sequences from Kansas, Nebraska and South Dakota showed 100% homology with 100% query cover to other P. maydis GenBank accessions (MG881848.1; OL342916.1; OL342915.1). Koch's postulates were not performed given the obligate nature of the pathogen (Muller and Samuels 1984). This report documents the first confirmation of tar spot on corn in Kansas, Nebraska, and South Dakota (Great Plains).