Culturing Phakopsora pachyrhizi on Detached Leaves and Urediniospore Survival at Different Temperatures and Relative Humidities M. Twizeyimana, Department of Crop Sciences, University of Illinois at Urbana-Champaign; and G. L. Hartman, United States Department of Agriculture–Agricultural Research Service and Department of Crop Sciences, Univer- sity of Illinois, Urbana 61801 amended media (10,39,53) or on filter ABSTRACT paper (38) inside petri dishes has been Twizeyimana, M., and Hartman, G. L. 2010. Culturing Phakopsora pachyrhizi on detached successful for maintaining leaf pieces to leaves and urediniospore survival at different temperatures and relative humidities. Plant Dis. evaluate the host–pathogen interactions 94:1453-1460. (10,39,54). Detached-leaf culturing has also been useful to study host–pathogen Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important foliar diseases of interactions for other rust fungi such as soybean worldwide. In a series of experiments, multiple objectives were addressed to determine Puccinia coronata (24), Uromyces viciae- the (i) longevity of detached soybean leaves, (ii) reproductive capacity of uredinia on leaves fabae (18), P. sorghi (21), and non-rust inoculated and/or incubated on the abaxial versus adaxial side of the leaf, (iii) reproductive ca- pacity of uredinia and urediniospore germination when spores were harvested at regular intervals pathogens Mycosphaerella fijiensis (55) or all at once, and (iv) effect of temperature and relative humidity (RH) on urediniospore germi- and Phytophthora infestans (49). Some nation. A detached-leaf assay using agar medium amended with 6-benzylaminopurine performed advantages of detached-leaf resistance better in retarding leaf chlorosis than filter paper alone among five soybean genotypes. Among evaluation assays compared with whole- the three susceptible genotypes tested, detached leaves of cv. Williams 82 had the lowest level of plant assays include improved environ- leaf chlorosis and often allowed for the greatest urediniospore production and germination rate. mental and pathogen control (41). The use Temperature and RH played significant roles in survival of urediniospore as measured by germi- of a detached-leaf assay to evaluate soy- nation rates. Viable urediniospores were harvested from infected soybean leaves maintained at bean rust resistance was validated in a room temperature (23 to 24°C at 55 to 60% RH) for up to 18 days, whereas freshly harvested Nigerian study (53) that demonstrated that urediniospores that were desiccated for 12 h before being placed in vials and maintained at room the disease severity recorded in the de- temperature remained viable for up to 30 days. Urediniospore hydration was the major factor for tached-leaf evaluations was positively the dormancy reversion; thermal shock with hydration and no thermal shock with hydration correlated with that observed on entire treatments had consistently similar urediniospore germination rates. In the RH experiment, ured- plants in the greenhouse or field. iniospores harvested from inoculated leaf pieces maintained at 85% RH had the highest germi- Urediniospores of Phakopsora pachyr- nation rates compared with higher and lower RH. Improvement in P. pachyrhizi cultural tech- niques and understanding of urediniospore survival will enhance our knowledge of the pathogen hizi have a relatively short life compared biology, host-plant relationship, and conditions that favor the infection, reproduction, and sur- with spores of other fungi because of their vival of the pathogen. thin spore wall (29). It was reported that urediniospores freshly harvested from infected leaves, then placed inside tightly sealed plastic microtubes and maintained Soybean rust, caused by Phakopsora P. pachyrhizi was first reported from the at room temperature (25 ± 3°C), survived pachyrhizi Syd. & P. Syd., is a major dis- continental Unites States in 2004 in Lou- for up to 18 days (13). ease that limits soybean (Glycine max (L.) isiana (45), and has become established in Methods for short- and long-term fungal Merr.) production in many areas of the the southern United States along the Gulf preservation have been reported (13,36,48). world. P. pachyrhizi was first reported in of Mexico, overwintering on kudzu (38). Short-term maintenance of soybean rust Japan in 1902 (17) and, for many decades The overwintering of P. pachyrhizi on urediniospores is done with periodic trans- after 1902, it was limited to the Eastern hosts such as kudzu and the potential long- fer of the pathogen to fresh detached Hemisphere (9). Soybean rust was reported distance transport of urediniospores (23) leaves. The most suitable long-term stor- from the major soybean-producing areas in provide sources of inoculum for epidemics age for rust urediniospores has been in Africa between the late 1990s and early to develop in soybean each year. Although liquid nitrogen (12). Frozen urediniospores 2000 (2,25,31), Paraguay in 2001, Brazil yield losses in the United States have not are revived by heat-shock followed by in 2002 (59), and Argentina in 2003 (42). been extensive, losses of up to 80% have hydration treatments (8,13). been reported from experimental plots in Temperature, humidity, solar radiation, Taiwan (16) and up to 55% from experi- and other factors affect fungal survival and Corresponding author: G. L. Hartman ments conducted in Southern Africa, South the infection process (1,11,14). Most fungal E-mail: [email protected] America, and the United States (34,35). spores are extremely sensitive to short peri- Trade and manufacturers’ names are necessary to Like other rust fungi, P. pachyrhizi is an ods (i.e., hours) of intense sunlight (32,43). report factually on available data; however, the obligate parasite and requires living host Like other fungi, the viability of P. pachyr- USDA neither guarantees nor warrants the standard tissue to establish and cause infection. This hizi urediniospores at the field level or in the of the product, and the use of the name by USDA renders the in vitro manipulation (e.g. grow- atmosphere can be greatly affected by tem- implies no approval of the product to the exclusion ing the pathogen on culture media) difficult perature and relative humidity (RH). An of others that may also be suitable. (13). Axenic culture of a limited number of optimal temperature range of 15 to 26°C Accepted for publication 16 August 2010. rust fungi, including Cronartium fusiforme has previously been reported for uredinio- (19), Peridermium pini (40), and Puccinia spore germination (50), with germination spp. (30), has been successful. Attempts to rates decreasing following the exposure of doi:10.1094/ PDIS-02-10-0131 culture Phakopsora pachyrhizi have not urediniospores to temperatures of 28.5 to This article is in the public domain and not copy- 42.5°C (28). Also, work on the longevity of rightable. It may be freely reprinted with custom- been successful (58). ary crediting of the source. The American Phyto- In vivo culturing of P. pachyrhizi on de- P. pachyrhizi urediniospores during periods pathological Society, 2010. tached soybean leaves placed on agar- of solar radiation has been done (22). Plant Disease / December 2010 1453 A key requirement to successfully pre- lowed by a cycle of 14 and 10 h light (380 replications. Leaf pieces collected as pre- vent yield losses due to soybean rust is to µmol m–2 s–1) and darkness, respectively, viously described were uniformly sprayed understand more about the biology of P. inside a tissue chamber (Percival Scien- with 200 µl of a 1 × 104 urediniospore/ml pachyrhizi, the host–pathogen relationship, tific, Inc.) maintained at 23°C. Prior to suspension using an air brush paint sprayer and the epidemiology of the disease. Many incubation, dishes were placed inside zip (Paasche Airbrush Co., Taiwan) attached to studies have contributed to our knowledge bags (Webster Industries, Peabody, MA). an air compressor. Inoculated leaf pieces of the infection process (26,27), the host When uredinia erupted (9 to 13 days after placed on 1% WA medium amended with range (37,46), and conditions that favor the inoculation), urediniospores (10 to 30) BAP in petri dishes were incubated as infection, development, and dissemination from a single uredinium were picked using described. Three days after inoculation, of the pathogen (7,22,38). a sharp sterile needle under a dissecting leaf pieces were observed daily under a With the aim to improve methods of P. microscope at ×20 magnification and were dissecting microscope at ×20 magnifica- pachyrhizi isolation, preservation, uredin- mixed with 40 µl of sterile water. The tion to record days to lesion appearance, iospore increase, and techniques for inocu- urediniospore suspension was then spread days to uredinia formation and uredinia lation, and to increase understanding of the onto 1.5% WA medium with a sterile glass eruption, number of uredinia on the conditions that favor disease development, rod. Two hours later, a single germinated abaxial and adaxial side of leaf pieces, and multiple objectives were addressed to de- urediniospore was removed using a sharp uredinia per lesion. Uredinia produced on termine: (i) longevity of detached soybean sterile needle and transferred to a drop of the abaxial side were counted from a 1- leaves, (ii) reproductive capacity of ured- sterile water with 0.01% Tween 20 and cm2 leaf area whereas uredinia produced inia when leaves were inoculated and/or placed on a new detached leaf of Williams on the adaxial side were counted from the incubated using the abaxial versus adaxial 82 appressed on 1% WA amended with whole leaf piece because the numbers were side, (iii) reproductive capacity of uredinia BAP in a petri dish. A different sharp ster- so few. and urediniospore germination when har- ile needle was used for picking a new ger- Effect of harvest intervals on uredinia vested at regular intervals or all at once, minating urediniospore. All urediniospores spore production and urediniospore and (iv) effect of temperature and RH on resulting from a single uredinium that germination.